Indirect homogeneous mobility shift assays for the detection of biologics in patient samples

ABSTRACT

The present invention provides a sensitive and specific indirect homogeneous mobility shift assay using size exclusion chromatography to measure biologics such as vedolizumab and ustekinumab in a patient sample. The assays of the present invention are particularly advantageous for detecting the presence or level of biologics that target complex or large antigens including cell surface proteins, transmembrane proteins, heavily glycosylated proteins, and multimeric proteins, as well as antigens that cannot be purified, impure antigens, and partially or substantially purified antigens. The present invention also provides isolated soluble α4β7 integrin heterodimers and isolated soluble IL-12p40 monomers that are suitable for use in the indirect assays described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 15/603,137 filed May 23, 2017 which is a continuation of PCT/IB2015/059381, filed Dec. 4, 2015, which claims priority to U.S. Provisional Application No. 62/088,465, filed Dec. 5, 2014, U.S. Provisional Application No. 62/113,317, filed Feb. 6, 2015, and U.S. Provisional Application No. 62/158,791, filed May 8, 2015, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 8, 2019 is named 56884867302 SL.txt and is 40,507 bytes in size.

BACKGROUND OF THE INVENTION

Inflammatory bowel disease (IBD), which occurs world-wide and afflicts millions of people, is the collective term used to describe three gastrointestinal disorders of unknown etiology: Crohn's disease (CD), ulcerative colitis (UC), and indeterminate colitis (IC). IBD, together with irritable bowel syndrome (IBS), will affect one-half of all Americans during their lifetime, at a cost of greater than $2.6 billion dollars for IBD and greater than $8 billion dollars for IBS. A primary determinant of these high medical costs is the difficulty of diagnosing digestive diseases and how these diseases will progress. The cost of IBD and IBS is compounded by lost productivity, with people suffering from these disorders missing at least 8 more days of work annually than the national average.

Despite the successes of anti-TNFα therapies in the treatment of IBD, a subpopulation of patients are refractory to treatment, highlighting an unmet medical need for new therapies. Vedolizumab is a gut-specific, α4β7 integrin-neutralizing monoclonal antibody, which does not affect peripheral blood cell counts and appears to lack systemic effects. Vedolizumab is a new anti-inflammatory treatment option for the management of therapy-refractory patients. In addition, ustekinumab is a IL12p40 monoclonal antibody, which is another novel IBD therapeutic. However, the availability of diagnostic tests to accurately measure the levels of biologics such as vedolizumab and ustekinumab is necessary for the effective use of these novel therapeutics in IBD patients.

As such, there is a need in the art for assays to detect the presence or level of biologics such as vedolizumab and ustekinumab in a patient sample to monitor drug therapy and to guide treatment decisions. Such assays are particularly useful for the therapeutic management of diseases such as ulcerative colitis and Crohn's disease using an individualized approach to monitor drug efficacy and optimize therapy accordingly, and can include assessing disease course and clinical parameters such as pharmacodynamics, disease activity indices, disease burden, and inflammatory biomarkers. The present invention satisfies this need and provides related advantages as well.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel indirect homogeneous mobility shift assays for detecting and measuring the presence or level of a biologic in a sample. The assays of the present invention are particularly advantageous for detecting and measuring the presence or level of biologics that target complex antigens including cell surface proteins, transmembrane proteins, heavily glycosylated proteins, multimeric proteins, and the like. As such, the present invention provides information for guiding treatment decisions for those subjects receiving therapy with a biologic agent and improves the accuracy of optimizing therapy, reducing toxicity, and/or monitoring the efficacy of therapeutic treatment to biologic therapy. The present invention also provides isolated soluble α4β7 integrin heterodimers and isolated soluble IL-12p40 monomers that are suitable for use in the assays described herein.

In certain aspects, the present invention provides a method for determining the presence or level of a biologic in a sample, the method comprising:

(a) contacting the sample with an unlabeled soluble antigen that binds to the biologic to form an unlabeled complex between the antigen and the biologic in the sample;

(b) contacting the sample from step (a) with a labeled form of the biologic to form a labeled complex between the antigen and the labeled biologic;

(c) subjecting the unlabeled and labeled complexes to size exclusion chromatography to separate the unlabeled and labeled complexes from free labeled biologic and to detect an amount of the free labeled biologic; and

(d) comparing the amount of the free labeled biologic detected in step (c) to a standard curve of known amounts of the biologic, thereby determining the presence or level of the biologic in the sample.

In some embodiments, the biologic includes antibodies, antibody fragments, proteins, polypeptides, peptides, fusion proteins, multivalent binding proteins, antibody-drug conjugates, vaccines, nucleic acids, sugars, recombinant forms thereof, engineered forms thereof, and combinations thereof.

In certain embodiments, the antigen is a soluble version (e.g., a soluble fragment, variant, or monomeric form) of a membrane-bound protein, a glycosylated protein, a multimeric protein, an insoluble protein, a protein that is difficult to express or purify, and/or a large protein. In certain instances, the antigen is a soluble extracellular domain of a membrane-bound protein (e.g., a soluble cytokine receptor extracellular domain). In certain other instances, the antigen is a soluble homodimer or heterodimer comprising the extracellular domains of two membrane-bound proteins (e.g., a soluble integrin heterodimer). In yet other instances, the antigen is a soluble protein that does not multimerize and remains in monomeric form once isolated and/or purified (e.g., a soluble cytokine variant with one or more cysteine residues mutated to minimize or eliminate the formation of multimers).

In other embodiments, the sample is a whole blood, serum, or plasma sample, e.g., from a subject receiving biologic therapy. In preferred embodiments, the sample is serum. In particular embodiments, the subject has a disease or disorder such as, e.g., an autoimmune disease (e.g., rheumatoid arthritis), an inflammatory disease (e.g., inflammatory bowel disease (IBD) such as Crohn's disease (CD) or ulcerative colitis (UC)), or cancer.

In particular embodiments, the standard curve is generated by incubating the antigen and the labeled biologic with a (e.g., two-fold) serial dilution of known amounts of the biologic. In certain embodiments, the area under the curve (AUC) of the free labeled biologic is plotted against (e.g., the logarithm of) known amounts of the biologic obtained from the standard curve, and the level of the biologic in the sample is calculated by interpolation, e.g., based upon the size of the peak area of the free labeled biologic. In other embodiments, free label added to a stock solution of labeled biologic is used as a labeled biologic loading control. The ratio of the free labeled biologic to free label is plotted against known amounts of biologic.

In one particular embodiment, the presence and/or level of an anti-α4β7 integrin drug (e.g., vedolizumab) is determined with an indirect homogeneous mobility shift assay using size exclusion chromatography as described herein.

In another particular embodiment, the presence and/or level of an anti-IL12p40 drug (e.g., ustekinumab) is determined with an indirect homogeneous mobility shift assay using size exclusion chromatography as described herein.

In other embodiments, the presence and/or level of anti-drug antibodies (ADA) (e.g., autoantibodies including HACA, HAHA, etc.) that are generated against anti-α4β7 integrin drugs and anti-IL12p40 drugs as well as other biologics is determined with a homogeneous mobility shift assay as described in, e.g., U.S. Pat. Nos. 8,574,855 and 8,865,417, and U.S. Patent Publication Nos. 2014/0051184 and 2014/0141983, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

In other aspects, the present invention provides an isolated soluble α4 integrin polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO:1 or SEQ ID NO:3. In yet other aspects, the present invention provides an isolated soluble (37 integrin polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 2 or SEQ ID NO:4.

In particular embodiments, the present invention provides an isolated soluble α4β7 integrin heterodimer comprising:

-   -   (a) an α4 integrin polypeptide having an amino acid sequence         that has at least 80% identity to SEQ ID NO:1, wherein the α4         integrin polypeptide is linked to a first member of a binding         pair (e.g., SEQ ID NO:3), and     -   (b) a β7 integrin polypeptide having an amino acid sequence that         has at least 80% identity to SEQ ID NO:2, wherein the β7         integrin polypeptide is linked to a second member of the binding         pair (e.g., SEQ ID NO:4).

In certain other aspects, the present invention provides an isolated soluble IL-12p40 polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NOS:6, 7, 11, 12, or 13.

In particular embodiments, the unlabeled soluble antigen used in the indirect homogeneous mobility shift assays of the present invention comprise the isolated soluble α4β7 integrin heterodimers or isolated soluble IL-12p40 polypeptides described herein.

In further aspects, the present invention provides expression vectors encoding the soluble polypeptides described herein, host cells comprising the expression vectors, and methods for generating the soluble polypeptides described herein.

Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the principle of the indirect homogeneous mobility shift assays (HMSA) of the present invention. Using vedolizumab (“VLM” or “Vedo”) as a non-limiting example, in the first step, serum is added to a 96 well plate along with integrin α4β7 and diluent buffer. In the second step, labeled VLM (e.g., Vedo Alexa 488) is added. The samples are then injected sequentially on an HPLC size exclusion column.

FIG. 2 shows the chromatograms of Vedo Alexa 488 (left) and Vedo Alexa 488 plus integrin α4β7 antigen (right) with retention times (x-axis) and light units (y-axis) of one embodiment of the present invention. Both are in 4% normal human serum. Note that the antigen binds up the majority of the labeled VLM.

FIG. 3 shows the chromatograms from standard curve overlays with the retention time (x-axis) and light units (y-axis) of the various components of a VLM assay in one embodiment of the present invention. “Vedo Alexa 488/α4β7”=Alexa Fluor® 488-labeled VLM bound to a soluble α4β7 heterodimer. “Alexa 488”=Blocked (e.g., inactivated) Alexa Fluor® 488 loading control.” Note that the free Vedo Alexa 488 peak area gets larger when there is more therapeutic VLM present.

FIG. 4 shows a VLM standard curve in one embodiment of the present invention. The standard curve was generated using a serial dilution of VLM with a concentration range of between 0.15625 μg/ml and 80 μg/ml.

FIG. 5 shows a VLM drug assay validation in one embodiment of the present invention. The limit of blank (LOB), limit of detection (LOD), lower limit of quantitation (LLOQ), and upper limit of quantitation (ULOQ) were calculated using a standard curve generated from a serial dilution of VLM with a concentration range of between 0.15625 μg/ml and 80 μg/ml.

FIG. 6 shows the intra-assay precision and accuracy of the VLM drug assay in one embodiment of the present invention.

FIG. 7 shows the retention time (x-axis) and light units (y-axis) of an anti-vedolizumab autoantibody (ATV) assay in one embodiment of the present invention.

FIG. 8 shows the validation of the ATV assay in one embodiment of the present invention.

FIG. 9 shows the validation of the ATV assay in one embodiment of the present invention.

FIG. 10 shows the interassay precision of the ATV assay in one embodiment of the present invention.

FIG. 11 shows a ustekinumab (UTK) drug assay validation in one embodiment of the present invention. The limit of blank (LOB), limit of detection (LOD), lower limit of quantitation (LLOQ), and upper limit of quantitation (ULOQ) were calculated using a standard curve generated from a serial dilution of UTK with a concentration range of between 0.078 μg/ml and 40 μg/ml.

FIG. 12 shows the validation of the assay for autoantibodies to ustekinumab (ATU) in one embodiment of the present invention.

FIG. 13 shows a schematic diagram of the exemplary embodiments of the soluble α4 integrin antigen and soluble β7 integrin antigen of the present invention. Left: Full-length proteins. Right: Truncated α4β7 integrin heterodimer with a cysteine bridge of the ACID-BASE peptide.

FIGS. 14A and 14B show the linearity of drug dilution in human serum for (A) VLM and (B) autoantibodies to vedolizumab (ATV).

FIGS. 15A, 15B and 15C show an analysis of common interfering agents in serum: (A) hemolyzed serum interference; (B) lipemic serum interference; and (C) RF serum interference.

FIG. 16 shows the principle of the ustekinumab (UTK) homogeneous mobility shift assay (HMSA).

FIGS. 17A and 17B show the linearity of dilution in normal human serum for (A) UTK and (B) autoantibodies to ustekinumab (ATU).

FIGS. 18A, 18B and 18C show an analysis of common interfering agents in serum: (A) hemolyzed serum interference; (B) lipemic serum interference; and (C) RF serum interference.

FIG. 19 shows integrin α4β7 substrate interference in one embodiment of the VLM assay of the present invention.

FIG. 20 shows standard curves using fixed amounts of labeled vedolizumab (“Vedo488”) and integrin α4β7 antigen (top left), as well as 2-fold increased (top right) or 4-fold increased (bottom left) amounts of both reagents. Note that the top end of the curve saturates at higher VLM concentrations. Similarly, the bottom end flattens out at slightly higher levels of VLM.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based in part on the discovery that an indirect homogeneous mobility shift assay (HMSA) using size exclusion chromatography is particularly advantageous for measuring the presence or level of biologics that target antigens having one or more of the following characteristics: cell surface or membrane-bound, (heavily) glycosylated, multimeric (e.g., forms heterodimers, homodimers, etc.), insoluble, difficult to express, difficult to purify, large in size, and combinations thereof. In certain aspects, the use of a soluble form (e.g., a soluble fragment, variant, or monomer) of the antigen overcomes the difficulties and limitations associated with antigens having one or more of the above characteristics and enables the precise and accurate measurement of any biologic of interest in a sample from a patient receiving therapy with that biologic.

The principle behind the indirect assays of the present invention is that the amount of (unlabeled) biologic in a sample (e.g., serum) obtained from a patient receiving biologic therapy determines how much unlabeled antigen remains free to bind to a labeled form of the biologic. By tracking changes in the area of the free (unbound) labeled biologic, the presence or level of (unlabeled) biologic in the patient sample can be determined. More particularly, the relative amount (e.g., ratio) of labeled and unlabeled biologic determines how much antigen is bound to each and determines the amount (e.g., peak area) of free labeled biologic following size exclusion chromatography. The amount (e.g., peak area) of the free labeled biologic can then be compared to a standard curve of known amounts of the biologic to provide an accurate measurement of biologic levels in the patient sample with high sensitivity and dynamic range. In certain embodiments, the size of the peak area of free labeled biologic following size exclusion chromatography is calculated and compared to the standard curve to interpolate the concentration of biologic in a patient sample.

The importance of measuring serum concentrations of biologics is illustrated by the fact that the FDA requires pharmacokinetic and tolerability (e.g., immune response) studies to be performed during clinical trials. The present invention also finds utility in monitoring patients receiving these drugs to make sure they are getting the right dose, that the drug isn't being cleared from the body too quickly, and that they are not developing an immune response against the drug. Furthermore, the present invention is useful in guiding the switch between different drugs due to failure with the initial drug.

II. Definitions

As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

The terms “competition,” “competition-based,” and “indirect” are used interchangeably herein to refer to an assay of the present invention for determining the presence or level of (unlabeled) biologic in a sample that relies on detecting the amount of free (unbound) labeled biologic remaining in the sample after unlabeled antigen and labeled biologic are added (sequentially) to the sample.

The terms “VLM,” “VDZ,” and “Vedo” are used interchangeably herein to refer to vedolizumab.

The terms “biologic” or “biologic agent” or “biological drug” as used herein encompass products and substances produced from or extracted from a biological system (e.g., a living organism). Non-limiting examples of biologics include antibodies, antibody fragments, proteins, polypeptides, peptides, fusion proteins (e.g., Ig fusion proteins or Fc fusion proteins), multivalent binding proteins (e.g., DVD Ig), antibody-drug conjugates, vaccines, nucleic acids, sugars, recombinant forms thereof, engineered forms thereof, and combinations thereof.

The term “antibody” includes large (150 kDa) “Y-shaped” molecules that consist of two identical light chains (˜220 amino acids) and two identical heavy chains (˜440 amino acids) that are held together by a combination of covalent (disulfide) and non-covalent interactions. Each light and heavy chain consists of repeating segments of constant or variable regions that contain one intrachain disulfide bond. The variable regions are located at the N-termini of the light and heavy chains, while the constant domains are located at the C-termini of the light and heavy chains. The N-termini of the light and heavy chains come together to form the antigen-binding site. The light chain is comprised of one variable domain and one constant domain and the heavy chain is comprised of one variable domain and three constant domains. Located at the ends of the “Y” are two identical (bivalent) antigen-binding sites. The distance between the two antigen binding sites varies due to the flexible hinge region, and as a result, the antigen binding efficiency can be greatly increased. The formation of the antigen-binding region is caused by the pairing of the variable region from the heavy chain (V_(H)) with the variable region of the light chain (V_(L)). Variations in amino acid sequences of the variable regions are responsible for the vast diversity of antigen-binding sites, and the greatest variability occurs throughout three hypervariable regions, termed complementary determining regions (CDRs). The tail region of the antibody, known as the F_(C) region, is comprised of two constant domains (C_(H)2, and C_(H)3) from each of the heavy chains. The F_(C) region is responsible for recruiting effector functions through binding of F_(C) receptors on neutrophils and macrophages.

The term “antigen” includes any molecule, agent, or substance that (e.g., specifically) binds to or interacts with a biologic. As one non-limiting example, the antigen comprises a soluble fragment, variant, or monomer of a membrane-bound protein, a glycosylated protein, a multimeric protein, an insoluble protein, a protein that is difficult to express or purify, and/or a large protein. As another non-limiting example, the antigen comprises a soluble fragment of a cell surface molecule such an integrin receptor (e.g., α4β7 integrin), wherein the soluble fragment contains one or more extracellular domains of the corresponding full-length molecule (e.g., a soluble α4β7 antigen heterodimer comprising extracellular domain sequences from the corresponding full-length α4 and β7 proteins). As yet another non-limiting example, the antigen comprises a cytokine such as TNFα or a subunit thereof such as IL-12p40 that does not form homodimers or heterodimers.

The term “size exclusion chromatography” or “SEC” includes a chromatographic method in which molecules in solution are separated based on their size and/or hydrodynamic volume. It is applied to large molecules or macromolecular complexes such as proteins and their conjugates. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel filtration chromatography.

The term “complex” includes an antigen bound (e.g., by non-covalent means) to a biologic (e.g., an unlabeled or labeled biologic), and a biologic (e.g., a labeled biologic) bound (e.g., by non-covalent means) to an autoantibody against the biologic.

As used herein, an entity that is modified by the term “labeled” includes any antigen, molecule, protein, enzyme, antibody, antibody fragment, cytokine, or related species that is conjugated with another molecule or chemical entity that is empirically detectable. Chemical species suitable as labels include, but are not limited to, fluorescent dyes, e.g. Alexa Fluor® dyes such as Alexa Fluor® 488, quantum dots, optical dyes, luminescent dyes, and radionuclides, e.g., ¹²⁵I.

The phrase “fluorescence label detection” includes a means for detecting a fluorescent label. Means for detection include, but are not limited to, a spectrometer, a fluorimeter, a photometer, and a detection device commonly incorporated with a chromatography instrument such as, but not limited to, size exclusion-high performance liquid chromatography, such as, but not limited to, an Agilent-1200 HPLC System.

The term “subject,” “patient,” or “individual” typically includes humans, but also includes other animals such as, e.g., other primates, rodents, canines, felines, equines, ovines, porcines, and the like.

The term “sample” includes any biological specimen obtained from an individual. Samples include, without limitation, whole blood, plasma, serum, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells (PBMC), polymorphonuclear (PMN) cells), ductal lavage fluid, nipple aspirate, lymph (e.g., disseminated tumor cells of the lymph node), bone marrow aspirate, saliva, urine, stool (i.e., feces), sputum, bronchial lavage fluid, tears, fine needle aspirate (e.g., harvested by random periareolar fine needle aspiration), any other bodily fluid, a tissue sample such as a biopsy of a site of inflammation (e.g., needle biopsy), cellular extracts thereof, and an immunoglobulin enriched fraction derived from one or more of these bodily fluids or tissues. In some embodiments, the sample is whole blood, a fractional component thereof such as plasma, serum, or a cell pellet, or an immunoglobulin enriched fraction thereof. One skilled in the art will appreciate that samples such as serum samples can be diluted prior to the analysis. In certain embodiments, the sample is obtained by isolating PBMCs and/or PMN cells using any technique known in the art. In certain other embodiments, the sample is a tissue biopsy such as, e.g., from a site of inflammation such as a portion of the gastrointestinal tract or synovial tissue.

The term “isolated,” when applied to a nucleic acid or polypeptide, denotes that the nucleic acid or polypeptide is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term “purified” denotes that a nucleic acid or polypeptide gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or polypeptide is at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, or at least about 99% pure.

The term “soluble,” in the context of a polypeptide, refers to polypeptide that can be prepared in a soluble and functional form using a host cell or a cell-free protein synthesis system. For instance, a soluble polypeptide does not form insoluble aggregates comprising misfolded and/or functionally inactive polypeptides.

The term “nucleic acid” or “polynucleotide” includes deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to include a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, truncated forms, or fragments thereof, wherein the amino acid residues are linked by covalent peptide bonds.

The term “amino acid” refers to naturally-occurring and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

Amino acids may be referred to herein by either their name, their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Additionally, nucleotides may be referred to by their commonly accepted single-letter codes.

The steps of the methods of the present invention do not necessarily have to be performed in the particular order in which they are presented. A person of ordinary skill in the art would understand that other orderings of the steps of the methods of the invention are encompassed within the scope of the present invention.

Brackets, “[ ]” indicate that the species within the brackets are referred to by their concentration.

III. Description of the Embodiments

The present invention provides novel indirect homogeneous mobility shift assays for detecting and measuring the presence or level of a biologic in a sample. The assays of the present invention are particularly advantageous for detecting the presence or level of biologics that target complex or large antigens including cell surface proteins, transmembrane proteins, heavily glycosylated proteins, and multimeric proteins, as well as antigens that cannot be purified, impure antigens, and partially or substantially purified antigens. The present invention also provides isolated soluble α4β7 integrin heterodimers and isolated soluble IL-12p40 monomers that are suitable for use in the assays described herein.

In one aspect, the present invention provides a method for determining the presence or level of a biologic in a sample, the method comprising:

(a) contacting the sample with an unlabeled soluble antigen that binds to the biologic to form an unlabeled complex (e.g., a plurality of unlabeled complexes) between the antigen and the biologic in the sample;

(b) contacting the sample from step (a) with a labeled form of the biologic (“labeled biologic”) to form a labeled complex (e.g., a plurality of labeled complexes) between the antigen and the labeled biologic;

(c) subjecting the (e.g., plurality of) unlabeled and labeled complexes to size exclusion chromatography to separate the (e.g., plurality of) unlabeled and labeled complexes from free labeled biologic and to detect an amount of the free labeled biologic; and

(d) comparing the amount of the free labeled biologic detected in step (c) to a standard curve of known amounts of the biologic, thereby determining the presence or level of the biologic in the sample.

In some embodiments, the biologic includes antibodies, antibody fragments, proteins, polypeptides, peptides, fusion proteins, multivalent binding proteins, antibody-drug conjugates, vaccines, nucleic acids, sugars, recombinant forms thereof, engineered forms thereof, and combinations thereof. In particular embodiments, the biologic comprises an antibody (e.g., a monoclonal antibody) or a fragment thereof (e.g., an antigen-binding fragment of a monoclonal antibody) or a conjugate thereof (e.g., an antibody-drug conjugate). Non-limiting examples of antibody-based biologics are shown in Table 1.

In particular embodiments, the method of the present invention detects the presence of and/or measures the level of unbound (free) biologic in a sample, e.g., the population of biologic in a sample that is not bound to its (endogenous) target antigen or a fragment thereof.

In certain embodiments, the antigen is a soluble version (e.g., a soluble fragment, variant, or monomeric form) of a membrane-bound protein, a (heavily) glycosylated protein, a multimeric protein, an insoluble protein, a protein that is difficult to express or purify, and/or a large protein. In certain instances, the antigen is a soluble extracellular domain of a membrane-bound protein (e.g., a soluble cytokine receptor extracellular domain). In certain other instances, the antigen is a soluble homodimer or heterodimer comprising the extracellular domains of two membrane-bound proteins (e.g., a soluble integrin heterodimer). In yet other instances, the antigen is a soluble protein that does not multimerize and remains in monomeric form once isolated and/or purified (e.g., a soluble cytokine variant with one or more cysteine residues mutated to minimize or eliminate the formation of multimers).

In some embodiments, the antigen is a soluble fragment (e.g., extracellular domain) of a cell surface molecule such as, e.g., a cell adhesion molecule (CAM). Non-limiting examples of CAMs include immunoglobulin superfamily (IgSF) CAMs, integrins, cadherins, and selectins.

IgSF CAMs are any of a variety of polypeptides or proteins located on the surface of a cell that have one or more immunoglobulin-like fold domains, and which function in intercellular adhesion and/or signal transduction. In many cases, IgSF CAMs are transmembrane proteins. Non-limiting examples of IgSF CAMs include mucosal addressin cell adhesion molecule 1(MADCAM1), neural cell adhesion molecules (NCAMs; e.g., NCAM-120, NCAM-125, NCAM-140, NCAM-145, NCAM-180, NCAM-185, etc.), intercellular adhesion molecules (ICAMs, e.g., ICAM-1, ICAM-2, ICAM-3, ICAM-4, and ICAM-5), vascular cell adhesion molecule-1 (VCAM-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), L1 cell adhesion molecule (L1CAM), cell adhesion molecule with homology to L1 CAM (close homolog of L1) (CHL1), sialic acid binding Ig-like lectins (SIGLECs; e.g., SIGLEC-1, SIGLEC-2, SIGLEC-3, SIGLEC-4, etc.), nectins (e.g., Nectin-1, Nectin-2, Nectin-3, etc.), and nectin-like molecules (e.g., Nec1-1, Nec1-2, Nec1-3, Nec1-4, and Nec1-5.

Integrins are transmembrane αβ heterodimers and at least 18 α and eight β subunits are known in humans, generating 24 heterodimers. The α and β subunits have distinct domain structures, with extracellular domains from each subunit contributing to the ligand-binding site of the heterodimer. Non-limiting examples of integrins include α₁β₁, α₂β₁, α₃β₁, α₄β₁, α₅β₁, α₆β₁, α₇β₁, α₈β₁, α₉β₁, α₁₀β₁, α₁₁β₁, α_(v)β₁, α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈, α_(IIb)β₃, α₄β₇, β_(E)β₇, α₆β₄, α_(L)β₂, α_(M)β₂, α_(X)β₂, and α_(D)β₂.

In particular embodiments, the antigen is an α4β7 integrin and the biologic is an anti-α4β7 integrin drug such as vedolizumab (VLM). In certain instances, the soluble fragment of the α4β7 integrin that binds to the anti-α4β7 integrin drug comprises an α4 fragment comprising an amino acid sequence having at least 80% identity to SEQ ID NO:1 or SEQ ID NO:3 and/or a β7 fragment comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 2 or SEQ ID NO:4.

In other embodiments, the antigen is an α4β1 integrin and the biologic is an anti-α4β1 integrin drug such as natalizumab. In certain instances, the soluble fragment of the α4β1 integrin that binds to the anti-α4β1 integrin drug comprises a heterodimer of the extracellular domains of the α4 and β1 subunits.

Cadherins are calcium-dependent transmembrane proteins that play important roles in cell adhesion, forming adherens junctions to bind cells within tissues together. Non-limiting examples of cadherins include E-cadherin, N-cadherin, N-cadherin 2, and P-cadherin.

Selectins are heterophilic CAMs that bind fucosylated carbohydrates, e.g., mucins. The three family members are E-selectin (endothelial), L-selectin (leukocyte), and P-selectin (platelet).

In other embodiments, the antigen is a soluble fragment (e.g., extracellular domain) of a cell surface molecule such as, e.g., a cytokine receptor.

Non-limiting examples of cytokine receptors include type I cytokine receptors, type II cytokine receptors, members of the immunoglobulin (Ig) superfamily, tumor necrosis factor receptors, chemokine receptors, and TGFβ receptors. Examples of type I cytokine receptors include, but are not limited to, interleukin receptors (e.g., IL-2 receptor, IL-3 receptor, IL-4 receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL-9 receptor, IL-11 receptor, IL-12 receptor, IL-13 receptor, IL-15 receptor, IL-21 receptor, IL-23 receptor, IL-27 receptor, etc.), colony stimulating factor receptors (e.g., erythropoietin receptor, GM-CSF receptor, G-CSF receptor, etc.), hormone receptors or neuropeptide receptors (e.g., growth hormone receptor, prolactin receptor, etc.), and other cytokine receptors such as oncostatin M receptor and leukemia inhibitory factor receptor. Examples of type II cytokine receptors include, but are not limited to, interferon receptors (e.g., interferon-alpha/beta receptor, interferon-gamma receptor, etc.) and interleukin receptors (e.g., IL-10 receptor, IL-20 receptor, IL-22 receptor, IL-28 receptor, etc.). Examples of immunoglobulin (Ig) superfamily receptors include, but are not limited to, IL-1 receptor, CSF1, c-kit receptor, and IL-18 receptor. Examples of tumor necrosis factor receptors include, but are not limited to, TNF receptor (CD120), lymphotoxin β receptor, CD134, CD40, FAS, TNFRSF6B, CD27, CD30, CD137, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, RANK, osteoprotegerin, TNFRSF12A, TNFRSF13B, TNFRSF13 C, TNFRSF14, nerve growth factor receptor, TNFRSF17, TNFRSF18, TNFRSF19, TNFRSF21, TNFRSF25, and ectodysplasin A2 receptor. Examples of chemokine receptors include, but are not limited to, CXC chemokine receptors, CC chemokine receptors, C chemokine receptors, and CX3C chemokine receptors. Examples of TGFβ receptors include, but are not limited to, TGFβ receptor 1, TGFβ receptor 2, and TGFβ receptor 3.

In certain embodiments, the antigen is an IL-6 receptor and the biologic is an anti-IL-6 receptor drug such as tocilizumab. In certain instances, the soluble fragment of the IL-6 receptor that binds to the anti-IL-6 receptor drug comprises an extracellular domain of the IL-6 receptor.

In yet other embodiments, the antigen is a soluble fragment (e.g., extracellular domain) of a cluster of differentiation (CD) molecule. Non-limiting examples of CD molecules include CD3, CD4, CD8, CD11a, CD11b, CD14, CD15, CD16, CD19, CD20, CD22, CD24, CD25, CD30, CD31, CD34, CD38, CD45, CD56, CD61, CD91, CD114, CD117, CD182, and the like. In certain instances, the biologic that binds to a soluble fragment of a CD molecule is a member selected from the group consisting of visilizumab, priliximab, rituximab, ofatumumab, obinutuzumab, ibritumomab tiuxetan, tositumomab, ocrelizumab, veltuzumab, daclizumab, and combinations thereof.

In some embodiments, the antigen is a cytokine or a monomer thereof (e.g., a soluble cytokine variant with one or more cysteine residues mutated to minimize or eliminate the formation of multimers).

Non-limiting examples of cytokines include TNFα, TNF-related weak inducer of apoptosis (TWEAK), osteoprotegerin (OPG), IFN-α, IFN-β, IFN-γ, interleukins (e.g., IL-1α, IL-1β, IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-5, IL-6, soluble IL-6 receptor (sIL-6R), IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-23, and IL-27), adipocytokines (e.g., leptin, adiponectin, resistin, active or total plasminogen activator inhibitor-1 (PAI-1), visfatin, and retinol binding protein 4 (RBP4)), and the like.

In particular embodiments, the cytokine is a p40 subunit of IL-12 or IL-23 and the biologic is an anti-IL-12p40 drug such as ustekinumab (UTK). In certain instances, the cytokine is a p40 variant which comprises one or more cysteine residues mutated to minimize or eliminate the formation of multimers. In some instances, the p40 variant comprises an amino acid sequence having at least 80% identity to SEQ ID NOS:6, 7, 11, 12 or 13.

In other embodiments, the cytokine is TNFα and the biologic is an anti-TNFα drug. Non-limiting examples of anti-TNFα drugs include REMICADE® (infliximab), HUMIRA® (adalimumab), ENBREL® (etanercept), CIMZIA® (certolizumab pegol), SIMPONI® (golimumab), and combinations thereof.

The soluble antigens described herein can be produced by any method known to one of ordinary skill in the art, such as but not limited to, synthetic methods, such as solid phase and liquid phase synthesis, or recombinant biology methods.

In some embodiments, the sample is a whole blood, serum, or plasma sample, e.g., obtained from a subject receiving biologic therapy. In preferred embodiments, the sample is serum. In particular embodiments, the subject has a disease or disorder such as, e.g., an autoimmune disease (e.g., rheumatoid arthritis), an inflammatory disease (e.g., inflammatory bowel disease (IBD) such as Crohn's disease (CD) or ulcerative colitis (UC)), or cancer.

In particular embodiments, the standard curve is generated by incubating the antigen and the labeled biologic with a (e.g., two-fold) serial dilution of known amounts of the biologic. In certain embodiments, the area under the curve (AUC) of the free labeled biologic is plotted against (e.g., the logarithm of) known amounts of the biologic obtained from the standard curve, and the level of the biologic in the sample is calculated by interpolation, e.g., based upon the size of the peak area of the free labeled biologic. In other embodiments, a ratio of the free labeled biologic to a loading control (e.g., free label) is determined and used to normalize the level of the biologic in the sample from the standard curve.

In certain embodiments, the size exclusion chromatography (SEC) is size exclusion-high performance liquid chromatography (SE-HPLC). In particular embodiments, the (e.g., plurality of) unlabeled and labeled complexes are eluted first through a stationary phase, followed by the free labeled biologic. The underlying principle of SEC is that molecules or complexes of different sizes will elute (filter) through a stationary phase at different rates. This results in the separation of a solution of molecules or complexes based on size. Provided that all the molecules or complexes are loaded simultaneously or near simultaneously, molecules or complexes of the same size elute together. Each size exclusion column has a range of molecular weights that can be separated. The exclusion limit defines the molecular weight at the upper end of this range and is where molecules or complexes are too large to be trapped in the stationary phase. The permeation limit defines the molecular weight at the lower end of the range of separation and is where molecules or complexes of a small enough size can penetrate into the pores of the stationary phase completely and all molecules or complexes below this molecular mass are so small that they elute as a single band.

In some instances, the eluent is collected in constant volumes, or fractions. The more similar the molecules or complexes are in size, the more likely they will be in the same fraction and not detected separately. Preferably, the collected fractions are examined by spectroscopic techniques to determine the concentration of the molecules or complexes eluted. Typically, the spectroscopy detection techniques useful in the present invention include, but are not limited to, fluorometry, refractive index (RI), and ultraviolet (UV). In certain instances, the elution volume decreases roughly linearly with the logarithm of the molecular hydrodynamic volume (i.e., heaver molecules or complexes come off first).

A biologic (e.g., therapeutic antibody) can be labeled with any of a variety of detectable group(s). In certain embodiments, a biologic is labeled with a fluorophore or a fluorescent dye. In other embodiments, a biologic is labeled with a luminescent tag, a metal, a radionuclide, and the like. Specific immunological binding of an antigen to a labeled biologic or the amount of free labeled biologic can be detected directly or indirectly. A signal from the direct or indirect label can be analyzed, e.g., using a spectrophotometer to detect color from a chromogenic substrate, a radiation counter to detect radiation such as a gamma counter for detection of ¹²⁵I, or a fluorometer to detect fluorescence in the presence of light of a certain wavelength.

Non-limiting examples of fluorophores or fluorescent dyes include those listed in the Molecular Probes Catalogue, which is herein incorporated by reference (see, R. Haugland, The Handbook-A Guide to Fluorescent Probes and Labeling Technologies, 10^(th) Edition, Molecular probes, Inc. (2005)). Such exemplary fluorophores or fluorescent dyes include, but are not limited to, Alexa Fluor® dyes such as Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 635, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, and/or Alexa Fluor® 790, as well as other fluorophores including, but not limited to, Dansyl Chloride (DNS-Cl), 5-(iodoacetamida)fluoroscein (5-IAF), fluoroscein 5-isothiocyanate (FITC), tetramethylrhodamine 5- (and 6-)isothiocyanate (TRITC), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), 7-nitrobenzo-2-oxa-1,3,-diazol-4-yl chloride (NBD-Cl), ethidium bromide, Lucifer Yellow, 5-carboxyrhodamine 6G hydrochloride, Lissamine rhodamine B sulfonyl chloride, Texas Red™ sulfonyl chloride, BODIPY™, naphthalamine sulfonic acids (e.g., 1-anilinonaphthalene-8-sulfonic acid (ANS), 6-(p-toluidinyl)naphthalen-e-2-sulfonic acid (TNS), and the like), Anthroyl fatty acid, DPH, Parinaric acid, TMA-DPH, Fluorenyl fatty acid, fluorescein-phosphatidylethanolamine, Texas Red-phosphatidylethanolamine, Pyrenyl-phophatidylcholine, Fluorenyl-phosphotidylcholine, Merocyanine 540,1-(3-sulfonatopropyl)-4-[β-[2[(di-n-butylamino)-6 naphthyl]vinyl]pyridinium betaine (Naphtyl Styryl), 3,3′ dipropylthiadicarbocyanine (diS-C₃-(5)), 4-(p-dipentyl aminostyryl)-1-methylpyridinium (di-5-ASP), Cy-3 Iodo Acetamide, Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate, rhodamine 800, IR-125, Thiazole Orange, Azure B, Nile Blue, Al Phthalocyanine, Oxaxine 1, 4′, 6-diamidino-2-phenylindole (DAPI), Hoechst 33342, TOTO, Acridine Orange, Ethidium Homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE), Fura-2, Calcium Green, Carboxy SNARF-6, BAPTA, coumarin, phytofluors, Coronene, metal-ligand complexes, IRDye® 700DX, IRDye® 700, IRDye® 800RS, IRDye® 800CW, IRDye® 800, Cy5, Cy5.5, Cy7, DY 676, DY680, DY682, DY780, and mixtures thereof. Additional suitable fluorophores include enzyme-cofactors; lanthanide, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, or mutants and derivatives thereof.

Typically, the fluorescent group is a fluorophore selected from the category of dyes comprising polymethines, pthalocyanines, cyanines, xanthenes, fluorenes, rhodamines, coumarins, fluoresceins and BODIPY™.

In certain embodiments, the fluorescent group is a near-infrared (NIR) fluorophore that emits in the range of between about 650 to about 900 nm. Use of near infrared fluorescence technology is advantageous in biological assays as it substantially eliminates or reduces background from auto fluorescence of biosubstrates. Another benefit to the near-IR fluorescent technology is that the scattered light from the excitation source is greatly reduced since the scattering intensity is proportional to the inverse fourth power of the wavelength. Low background fluorescence and low scattering result in a high signal to noise ratio, which is essential for highly sensitive detection. Furthermore, the optically transparent window in the near-IR region (650 nm to 900 nm) in biological tissue makes NIR fluorescence a valuable technology for in vivo imaging and subcellular detection applications that require the transmission of light through biological components. Within aspects of this embodiment, the fluorescent group is preferably selected form the group consisting of IRDye® 700DX, IRDye® 700, IRDye® 800RS, IRDye® 800CW, IRDye® 800, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Alexa Fluor® 790, Cy5, Cy5.5, Cy7, DY 676, DY680, DY682, and DY780. In certain embodiments, the near infrared group is IRDye® 800CW, IRDye® 800, IRDye® 700DX, IRDye® 700, or Dynomic DY676.

Fluorescent labeling can be accomplished using a chemically reactive derivative of a fluorophore. Common reactive groups include amine reactive isothiocyanate derivatives such as FITC and TRITC (derivatives of fluorescein and rhodamine), amine reactive succinimidyl esters such as NHS-fluorescein, and sulfhydryl reactive maleimide activated fluors such as fluorescein-5-maleimide, many of which are commercially available. Reaction of any of these reactive dyes with a biologic results in a stable covalent bond formed between a fluorophore and a biologic.

In certain instances, following a fluorescent labeling reaction, it is often necessary to remove any non-reacted fluorophore from the labeled target molecule. This is often accomplished by size exclusion chromatography, taking advantage of the size difference between fluorophore and labeled protein.

Reactive fluorescent dyes are available from many sources. They can be obtained with different reactive groups for attachment to various functional groups within the target molecule. They are also available in labeling kits that contain all the components to carry out a labeling reaction. In certain instances, Alexa Fluor® 488 NHS ester is used from Life Technologies (Cat. No. A-10235).

IV. Indirect Homogeneous Mobility Shift Assays

The present invention provides novel indirect assays for detecting and measuring the presence or level of a biologic (“drug”) in a sample using size exclusion chromatography. The assays of the present invention are particularly advantageous for detecting the presence or level of drugs that target complex or large antigens including cell surface proteins, transmembrane proteins, heavily glycosylated proteins, and multimeric proteins, as well as antigens that cannot be purified, impure antigens, and partially or substantially purified antigens. The antigens are not labeled and thus patient drug/antigen complexes do not appear in the chromatogram. The principle behind the indirect assays is that the amount of patient drug determines how much unlabeled antigen remains free to bind to a labeled version of the drug. By tracking changes in the area of the free (unbound) labeled drug, one can determine how much patient drug is present.

In certain aspects, the first step of the indirect assays described herein comprises incubating a sample (e.g., serum) containing therapeutic drug (e.g., vedolizumab (VDZ)) with a fixed amount of antigen to the drug (e.g., soluble α4β7). In the second step, a fixed amount of labeled drug (e.g., VDZ coupled with Alexa Fluor® 488) is added. The amount of therapeutic drug in the sample determines how much antigen remains free and available to bind the labeled drug. This, in turn, determines how much labeled drug is free. Since the peak area of the free labeled drug is proportional to the amount of therapeutic drug in the sample, one can quantify the amount of therapeutic drug by interpolation against a standard curve containing known amounts of drug.

The following description of the principles of the indirect assays of the invention uses vedolizumab (VDZ) as the therapeutic drug for illustrative purposes only (see, FIG. 1), and is not intended to limit the scope of the assay methodology for detecting or measuring the presence or level of other biologics in patient samples:

-   -   1. To each patient sample (e.g., serum), fixed amounts of         antigen (e.g., soluble α4β7) and labeled VDZ are added. The         amount of antigen and labeled VDZ can be added to each sample in         a controlled ratio. For example, adding an amount of antigen         which would bind up about 75-80% of the labeled VDZ provides         optimal sensitivity without limiting the dynamic range of the         assay. The ratio of antigen to labeled VDZ was determined by         titrating the antigen with a fixed amount of labeled VDZ so that         when the antigen is added to the labeled VDZ, the peak of free         labeled VDZ is reduced by about 75-80% (see, FIG. 2).     -   2. Quantification can be performed by tracking the increase of         the labeled VDZ peak area (R_(t)=7.5-8.5 min). This area is         proportional to the amount of therapeutic drug present.         Tris-blocked Alexa488 can be added to labeled VDZ stock         solutions as a loading control. Raw chromatograms can be         collected in Agilent ChemStation and then exported to the         program “R” for automated analysis. The standard curve can be         generated by plotting the labeled VDZ peak area as a function of         the log of known VDZ sample concentrations. A 10-point standard         curve can be used and fitted with a 5-parameter logistic (5-PL)         model to account for asymmetry. Unknowns can be determined from         the standard curve by interpolation.

In certain embodiments, the ratio of antigen to labeled drug that is added to a sample is an amount of each reagent that provides the best compromise between the low-end sensitivity needed as well as a dynamic range that enables the measurement of drug in patient samples without requiring dilutions. As a non-limiting example, the ratio of antigen to labeled drug that is added to a sample is an amount of antigen that binds up about 75% to about 80% (e.g., about 75%, 76%, 77%, 78%, 79%, or 80%) of the labeled drug. In some instances, the ratio of antigen to labeled drug that is added to a sample is an amount of antigen that binds up at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the labeled drug. In other instances, the ratio of antigen to labeled drug that is added to a sample is an amount of antigen that binds up about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, about 50% to about 80%, about 60% to about 80%, about 70% to about 80%, about 50% to about 70%, about 60% to about 70%, or about 50% to about 60% of the labeled drug. The ratio of antigen to labeled drug can be determined by titrating the antigen with a fixed amount of labeled drug so that when the antigen is added to the labeled drug, the peak of free labeled drug is reduced by a desired percent (e.g., about 75-80%).

In certain other embodiments, the dynamic range of the indirect assays described herein can be improved by proportionately increasing the amount of both antigen and labeled drug that is added to a sample. In some instances, the amount of both antigen and labeled drug can be about 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, or 10-fold more than a reference amount of antigen and labeled drug. As a non-limiting example, the reference amount of labeled VDZ can be about 75 ng and the increased amount of labeled VDZ can be about 120 ng (i.e., 1.6-fold more than the reference amount).

In some embodiments, the lower limit of quantitation (LLOQ) of the indirect assays described herein is about 0.125 μg/mL, 0.25 μg/mL, 0.375 μg/mL, 0.5 μg/mL, 0.625 μg/mL, 0.75 μg/mL, 0.875 μg/mL, 1 μg/mL, 1.25 μg/mL, 1.5 μg/mL, 1.75 μg/mL, 2 μg/mL, 3 μg/mL, 4 μg/mL, or 5 μg/mL. In other embodiments, the upper limit of quantitation (ULOID) of the indirect assays described herein is about 8 μg/mL, 9 μg/mL, 10 μg/mL, 11 μg/mL, 12 μg/mL, 13 μg/mL, 14 μg/mL, 15 μg/mL, 16 μg/mL, 17 μg/mL, 18 μg/mL, 19 μg/mL, 20 μg/mL, 21 μg/mL, 22 μg/mL, 23 μg/mL, 24 μg/mL, 25 μg/mL, 26 μg/mL, 27 μg/mL, 28 μg/mL, 29 μg/mL, 30 μg/mL, 35 μg/mL, 40 μg/mL, 45 μg/mL, or 50 μg/mL. In particular embodiments, the LLOQ is about 1 μg/mL and the ULOQ is about 25 μg/mL.

V. Soluble α4β7 Integrin Polypeptide Antigens

In one aspect, the present invention provides an isolated soluble α4 integrin polypeptide comprising an amino acid sequence having at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 1. In some embodiments, the isolated soluble α4 integrin polypeptide comprises β-propeller repeats (i.e., repeats 1-7) and a thigh domain of the human α4 integrin extracellular domain (see, FIG. 13; “α4Δ620”), or a fragment thereof. In other embodiments, the isolated soluble α4 integrin polypeptide comprises β-propeller repeats, a thigh domain, and one or both Calf domains (i.e., Calf-1 and/or Calf-2) of the human α4 integrin extracellular domain, or a fragment thereof. In yet other embodiments, the isolated soluble α4 integrin polypeptide is a truncated receptor comprising the entire human α4 integrin extracellular domain. The isolated soluble α4 integrin polypeptide described herein includes a ligand binding domain or a portion thereof.

In some embodiments, the isolated soluble α4 integrin polypeptide also includes an ACID peptide. Such a peptide may have an amino acid sequence having at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:8. An ACID peptide can form an α-helical coiled-coil conformation with a BASE peptide. In some embodiments, the ACID peptide includes a cysteine residue that can form a disulfide bridge with a cysteine residue on the BASE peptide. Acid coiled-coil region peptides (ACID peptides) and basic coiled-coil region peptides (BASE peptides) are described in, e.g., O'Shea et al., Curr Biol, 1993, 3:658-667, Jun et al., Proc Natl Acad Sci U.S.A., 2001, 98(12):6830-6835, Takagi et al., Nat Struct. Biol., 2001, 8:412-416, Nishida et al., Immunity, 2006, 25:583-594, and Dong et al., Biochemistry, 2012, 51(44):8814-8828.

In some embodiments, the isolated soluble α4 integrin polypeptide includes a linker, such as one or more amino acid residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues. The linker can be located between the end of extracellular domain and the ACID peptide.

In some embodiments, the isolated soluble α4 integrin polypeptide comprises an amino acid sequence having at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:3.

The soluble α4 integrin polypeptide can also include an affinity tag or epitope tag, such as a histidine tag, avidin tag, V5 tag, FLAG tag, HA tag, Myc tag, cleavable tag, and the like. In some instances, the soluble α4 integrin polypeptide can include a fluorescent tag, such as GFP, DsRed, CFP, YFP, RFP, and the like, or other detectable tag, such as horseradish peroxidase, chloramphenicol acetyltransferase, beta-galactosidase, luciferase, and the like.

In another aspect, the present invention provides an isolated soluble β7 integrin polypeptide comprising an amino acid sequence having at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:2. In some embodiments, the isolated soluble β7 integrin polypeptide comprises a PSI domain, an I-like domain, hybrid domains, and an I-EGF 1 domain of the human β7 integrin extracellular domain (see, FIG. 13; “β7Δ527”), or a fragment thereof. In other embodiments, the isolated soluble β7 integrin polypeptide comprises one or more of the PSI domain, I-like domain, and one or both hybrid domains of the human β7 integrin extracellular domain, or a fragment thereof. In yet other embodiments, the isolated soluble β7 integrin polypeptide comprises a PSI domain, an I-like domain, hybrid domains, I-EGF domains (i.e., domains 1-4), and optionally a β-tail of the human β7 integrin extracellular domain, or a fragment thereof. In further embodiments, the isolated soluble β7 integrin polypeptide is a truncated receptor comprising the entire human β7 integrin extracellular domain. The soluble β7 integrin polypeptide described herein includes a ligand binding domain or a portion thereof.

In some embodiments, the isolated soluble β7 integrin polypeptide also includes a BASE peptide. Such a peptide may have an amino acid sequence having at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:9. A BASE peptide can form an α-helical coiled-coil conformation with an ACID peptide.

In some embodiments, the isolated soluble β7 integrin polypeptide includes a protease cleavage site. In some instances, the cleavage site is a tobacco etch virus (TEV) protease cleavage site. The TEV site can include the amino acid sequence EXXYXQ/S, wherein X is any amino acid residue (SEQ ID NO:10). The TEV site may be located upstream the BASE peptide.

In some embodiments, the isolated soluble β7 integrin polypeptide includes a linker, such as one or more amino acid residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue. The linker can be located between the end of the EGF-I domain of the β7 integrin extracellular domain and the cleavage site.

In some embodiments, the isolated soluble β7 integrin polypeptide further comprises an affinity tag or epitope tag. Useful affinity or epitope tags include, but are not limited to, a histidine tag, avidin tag, V5 tag, FLAG tag, HA tag, Myc tag, cleavable tag, and the like. In some instances, the soluble β7 integrin polypeptide can include a fluorescent tag, such as GFP, DsRed, CFP, YFP, RFP, and the like, or other detectable tag, such as horseradish peroxidase, chloramphenicol acetyltransferase, beta-galactosidase, luciferase, and the like.

In some embodiments, the isolated soluble β7 integrin polypeptide comprises an amino acid sequence having at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:4.

The isolated soluble β7 integrin polypeptide can associate with a soluble α4 integrin polypeptide described herein to form a complex, such as a covalently linked heterodimer. In some instances, the α4β7 integrin complex can bind to α4β7 ligands, such as but not limited to VCAM-1 and MAdCAM-1, and to antibodies directed against α4β7 integrin, such as, but not limited to, vedolizumab, natalizumab, and etrolizumab.

In some aspects, the present invention provides an isolated soluble α4β7 integrin polypeptide comprising a soluble α4 integrin polypeptide having an amino acid sequence that has at least 80% identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 1, wherein the α4 integrin polypeptide is linked to a first member of a binding pair, and a soluble β7 integrin polypeptide having an amino acid sequence that has at least 80% identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO: 2, wherein the (37 integrin polypeptide is linked to a second member of the binding pair.

In some embodiments, the binding pair can be any peptides, molecules, motifs, and compounds that can allow the α4 subunit and β7 subunit to form a heterodimer, such as a covalently linked heterodimer. The α4β7 heterodimer is capable of binding to α4β7 ligands, such as, but not limited to VCAM-1 and MAdCAM-1, and to antibodies directed against α4β7 integrin, such as, but not limited to, vedolizumab, natalizumab, and etrolizumab. The soluble α4 integrin polypeptide and the soluble β7 integrin polypeptide can heterodimerize via a cysteine bridge or a derivative thereof (see, FIG. 13). In some embodiments, the binding pair is selected from the group consisting of coiled-coil peptides, leucine zipper peptides, dock-and-lock peptides, avidin-biotin, and derivatives thereof. In some instances, the coiled-coil peptides are ACID-BASE peptides.

The polypeptides described herein can be produced by any method known to one of ordinary skill in the art, such as but not limited to, synthetic methods, such as solid phase and liquid phase synthesis, or recombinant biology methods, such as those described herein.

In other aspects, the present invention provides an expression vector encoding a soluble α4β7 integrin polypeptide comprising a first polynucleotide sequence comprising a nucleic acid sequence encoding a soluble α4 integrin polypeptide having an amino acid sequence that has at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:1 and a nucleic acid sequence encoding a first member of a binding pair, and a second polynucleotide sequence comprising a nucleic acid sequence encoding a soluble (37 integrin polypeptide having an amino acid sequence that has at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:2 and a nucleic acid sequence encoding a second member of the binding pair. In some embodiments, the first member of the binding pair is an ACID peptide. In some embodiments, the first member of the binding pair is a BASE peptide. In some instances, the second polynucleotide sequence further comprises a nucleic acid sequence encoding an affinity tag, such as a histidine tag. In some instances, the second polynucleotide sequence further comprises a nucleic acid sequence encoding a protease cleavage site, such as a TEV site.

In some embodiments, the first polynucleotide sequence comprises a nucleic acid sequence encoding a polypeptide having an amino acid sequence that has at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:3. In some embodiments, the second polynucleotide sequence comprises a nucleic acid sequence encoding a polypeptide having an amino acid sequence that has at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:4.

In some embodiments, the expression vector is capable of directing expression of the polynucleotide sequences preferentially in a particular cell type. The expression vector can be a plasmid, phage, phagemid, cosmid, bacteriophage, baculovirus vector, lentiviral vector, retroviral vector, adenoviral vector, yeast plasmid, and the like. The expression vector can also comprise a promoter. Useful promoters include constitutive promoters and inducible promoters. The first polynucleotide sequence and/or the second polynucleotide sequence of the expression vector may be operably linked to a promoter. The promoter can be selected depending on the host cell containing the expression vector or used to generate or produce the soluble α4β7 integrin polypeptide encoded by the expression vector described herein. The expression vector may include regulatory elements, a selectable marker cassette, antibiotic resistance cassette, or any other component that facilitates the expression of the polypeptide by a host cell.

In some embodiments, the first and second polynucleotide sequences are found in a single expression vector. Such polynucleotide sequence can be located in a bicistronic expression vector such that an IRES sequence is located between the first and second polynucleotides sequences in the vector. A single promoter can drive the expression of both polynucleotides sequences. In some embodiments, the first polynucleotide sequence is operably linked to the promoter and is located immediately upstream from a nucleic acid sequence encoding a ribosomal skip, such as a viral 2A peptide, which is immediately upstream of the second polynucleotide sequence. In other embodiments, the second polynucleotide sequence is operably linked to the promoter and is located immediately upstream from a nucleic acid sequence encoding a ribosomal skip which is immediately upstream of the first polynucleotide sequence. The soluble α4 integrin polypeptide and soluble β7 integrin polypeptide can be generated from one expression vector.

Methods for constructing an expression vector are known to those of ordinary skill in the art. Detailed descriptions of protocols and methods are described in, e.g., Green, M. R., and Sambrook, J., eds., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), Ausubel, F. M., et al., Current Protocols in Molecular Biology (Supplement 99), John Wiley & Sons, New York (2012); Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology: Volume 152, Academic Press, Inc., San Diego, Calif., (1987); and PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, Calif., (1990).

In other aspects, the present invention provides a host cell comprising any one of the expression vectors encoding a soluble α4β7 integrin polypeptide described herein. The host cell can be a stable cell line, such as, but not limited to, HEK293 cells, CHO cells, COS cells, Jurkat cells, NIH3T3 cells, and derivatives thereof. The host cell can be a bacterial cell, yeast cell, fungal cell, algal cell, plant cell, insect cell, animal cell, mammalian cell, non-human cell, or human cell. Suitable host cells are described in Goeddel, Gene Expression Technology: Methods in Enzymology, 185, Academic Press, San Diego, Calif., (1990).

The expression vector can be introduced into the host cell by methods including, but not limited to, transformation, transfection, lipofection, nucleofection, microinjection, electroporation, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:polynucleotide conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.

In yet another aspect, the present invention provides a method of generating a soluble α4β7 integrin polypeptide encoded by any one of the expression vectors described herein. The method comprises (a) introducing the expression vector encoding the soluble α4β7 integrin polypeptide into a host cell, (b) culturing the resulting host cell under conditions to produce the soluble α4β7 integrin polypeptide, and (c) isolating the soluble α4β7 integrin polypeptide.

The cells containing the expression vector can be cultured under conditions that allow, promote or induce the production of the soluble α4β7 integrin polypeptide.

The soluble α4 integrin polypeptide, soluble β7 integrin polypeptide and soluble α4β7 integrin polypeptide can be purified from, for example, a cell culture supernatant or soluble fraction of a cell extract, according to standard methods known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J. C. Janson and Lars Ryden, eds., VCH Publishers, New York, (1989)) to obtain substantially pure polypeptides. Methods for protein purification, chromatography, electrophoresis, centrifugation, and crystallization are described in, e.g., Coligan et al., Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York, (2000).

The soluble recombinant α4β7 integrin polypeptide can complex with its cognate ligand, such as a ligand that specifically binds to wild-type, full-length α4β7 integrin. The soluble recombinant α4β7 integrin polypeptide can be an antigen for an anti-α4β7 integrin antibody.

VI. Soluble IL-12p40 Polypeptide Antigens

In one aspect, the present invention provides an isolated soluble IL-12p40 polypeptide comprising an amino acid sequence having at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NOS:6, 11, 12, or 13. In some embodiments, the polypeptide further comprises an affinity tag. In other embodiments, the isolated soluble IL-12p40 polypeptide comprising an amino acid sequence having at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 8′7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:7. In some embodiments, the polypeptide further comprises an affinity tag. In particular embodiments, the soluble IL-12p40 polypeptide is a monomer, and cannot dimerize or form multimers.

In another aspect, the present invention provides an expression vector encoding a soluble IL-12p40 polypeptide comprising a polynucleotide sequence comprising a nucleic acid sequence encoding an IL-12p40 polypeptide having an amino acid sequence that has at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NOS:6, 11, 12, or 13. The polynucleotide sequence can further comprise a nucleic acid sequence encoding an affinity tag. Such an affinity tag can be a histidine tag, such as hexahistidine. Other non-limiting examples of affinity tags include an avidin tag, V5 tag, FLAG tag, HA tag, Myc tag, cleavable tag, and the like. In some embodiments, the polynucleotide comprising a nucleic acid sequence encoding an IL-12p40 polypeptide having an amino acid sequence that has at least 80% sequence identity, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to SEQ ID NO:7.

In some embodiments, the expression vector is capable of directing expression of the polynucleotide sequences preferentially in a particular cell type. The expression vector can be a plasmid, phage, phagemid, cosmid, bacteriophage, baculovirus vector, lentiviral vector, retroviral vector, adenoviral vector, yeast plasmid, and the like. The expression vector can also comprise a promoter. Useful promoters include constitutive promoters and inducible promoters. The polynucleotide sequence of the expression vector may be operably linked to a promoter. The promoter can be selected depending on the host cell selected to generate or produce the soluble IL-12p40 polypeptide encoded by the expression vector described herein. The expression vector may include regulatory elements, a selectable marker cassette, antibiotic resistance cassette, or any other component that facilitates the expression of the polypeptide.

Methods for constructing an expression vector are known to those of ordinary skill in the art. Detailed descriptions of protocols and methods are described in, e.g., Green, M. R., and Sambrook, J., eds., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012); Ausubel, F. M., et al., Current Protocols in Molecular Biology (Supplement 99), John Wiley & Sons, New York (2012); Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Volume 152, Academic Press, Inc., San Diego, Calif. (1987); and PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, Calif., (1990).

In other aspects, the present invention provides a host cell comprising any one of the expression vectors encoding a soluble IL-12p40 polypeptide described herein. The host cell can be a stable cell line, such as, but not limited to, HEK293 cells, CHO cells, COS cells, Jurkat cells, NIH3T3 cells, and derivatives thereof. The host cell can be a bacterial cell, yeast cell, fungal cell, algal cell, plant cell, insect cell, animal cell, mammalian cell, non-human cell, or human cell. Suitable host cells are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., 1990.

The expression vector can be introduced into the host cell by methods including, but not limited to, transformation, transfection, lipofection, nucleofection, microinjection, electroporation, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:polynucleotide conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.

In yet another aspect, the present invention provides method of generating a soluble IL-12p40 polypeptide encoded by any one of the expression vectors described herein. The method comprises (a) introducing the expression vector encoding the soluble IL-12p40 polypeptide into a host cell, (b) culturing the resulting host cell under conditions to produce the soluble IL-12p40 polypeptide, and (c) isolating the soluble IL-12p40 polypeptide.

The cells containing the expression vector can be cultured under conditions that allow, promote or induce the production of the soluble IL-12p40 polypeptide.

The soluble IL-12p40 polypeptide can be purified from, for example, a cell culture supernatant or soluble fraction of a cell extract, according to standard methods known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J. C. Janson and Lars Ryden, eds., VCH Publishers, New York, (1989)) to obtain substantially pure polypeptides. Methods for protein purification, chromatography, electrophoresis, centrifugation, and crystallization are described in, e.g., Coligan et al., Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York, (2000).

Unlike wild-type IL-12p40, the soluble recombinant IL-12p40 polypeptide described herein cannot form a dimer, trimer, or oligomer. In particular embodiments, the presence of either two cysteine to alanine substitutions (SEQ ID NO:6) or two cysteine to serine substitutions (SEQ ID NO:11) in the wild-type IL-12p40 polypeptide sequence prevents the IL-12p40 antigen from oligomerizing. In other embodiments, the soluble IL-12p40 polypeptide monomer has the wild-type IL-12p40 polypeptide sequence with one cysteine to alanine substitution and one cysteine to alanine substitution (SEQ ID NOS:12 or 13). An anti-IL-12p40 antibody, such as ustekinumab, can specifically bind to the soluble recombinant IL-12p40 polypeptide.

VII. Biologic Therapy

The indirect homogeneous mobility shift assays of the present invention are suitable for detecting and/or measuring the presence or level of a biologic in a sample from a subject (e.g., a subject receiving biologic therapy). Non-limiting examples of biologics include antibodies, antibody fragments, proteins, polypeptides, peptides, fusion proteins (e.g., Ig fusion proteins or Fc fusion proteins), multivalent binding proteins (e.g., DVD Ig), antibody-drug conjugates, vaccines, nucleic acids, sugars, recombinant forms thereof, engineered forms thereof, and combinations thereof.

Examples of antibody-based biologics include, but are not limited to, diagnostic or therapeutic monoclonal antibodies and antigen-binding fragments or conjugates thereof. In certain embodiments, the antibody comprises an anti-integrin drug such as an anti-α4β7 integrin drug (e.g., vedolizumab (ENTYVIO™), etrolizumab) and/or an anti-α4β1 integrin drug (e.g., natalizumab (TYSABRI®)). In other embodiments, the antibody comprises an anti-cytokine drug such as an anti-IL12p40 drug (e.g., ustekinumab))(STELARA®)). In yet other embodiments, the antibody comprises an anti-cytokine receptor drug such as an anti-IL-6 receptor drug (e.g., tocilizumab (ACTEMRA®)). In further embodiments, the antibody comprises an anti-CD receptor drug such as an anti-CD3 receptor drug (e.g., visilizumab), an anti-CD4 receptor drug (e.g., priliximab), an anti-CD20 receptor drug (e.g., rituximab (RITUXAN®), ofatumumab (ARZERRA®), obinutuzumab (GAZYVA®), ibritumomab tiuxetan (ZEVALIN®), tositumomab (BEXXAR®), ocrelizumab, veltuzumab, an anti-CD25 receptor drug (e.g., daclizumab (ZENAPAX®)), or combinations thereof. In other embodiments, the antibody comprises an anti-TNFα drug such as infliximab (REMICADE®), adalimumab (HUMIRA®), etanercept (ENBREL®), golimumab (SIMPONI®), certolizumab pegol (CIMZIA®), or combinations thereof. Additional examples of antibody-based biologics include antibody-drug conjugates such as brentuximab vedotin (ADCETRIS®).

Table 1 provides an exemplary and non-exhaustive list of diagnostic and therapeutic monoclonal antibodies which have either been approved or are currently in development. An extensive list of biologic medicines including monoclonal antibody-based therapeutics and diagnostics in clinical development and approved products is provided in the 2006 PhRMA Report entitled “418 Biotechnology Medicines in Testing Promise to Bolster the Arsenal Against Disease” and the 2013 PhRMA Report entitled “Medicines in Development—Biologics,” the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

TABLE 1 Monoclonal Antibodies (mAb) Product Name Company Indication Digestive Disorders ABT 874 Abbott Laboratories Crohn's disease AMG 139/MEDI-2070 Amgen Crohn's disease AMG 181/MEDI-7183 Amgen Crohn's disease, ulcerative colitis anrukinzumab (IMA-638) Pfizer ulcerative colitis anti-IP10 Bristol-Myers Squibb Crohn's disease, ulcerative colitis clazakizumab (anti-IL6) Bristol-Myers Squibb, Alder Crohn's disease Biopharmaceuticals etrolizumab (rhuMAb-07) Genentech ulcerative colitis GSK1070806 (IL-18 mAb) GlaxoSmithKline inflammatory bowel disease Humira ® (adalimumab) AbbVie Crohn's disease MDX-1100 Millennium Pharmaceuticals ulcerative colitis Nuvion ® (visilizumab) PDL BioPharma I.V. steroid-refractory ulcerative colitis and Crohn's disease PF-00547659 Pfizer Crohn's disease PF-04236921 Pfizer Crohn's disease QAX576 Novartis Pharmaceuticals Crohn's disease Remicade ® (infliximab) Janssen Biotech Crohn's disease SAR252067 (anti-LIGHT mAb) Sanofi US Crohn's disease, ulcerative colitis SAR339658 (VLA2 antagonist) Sanofi US inflammatory bowel disease Simponi ® (golimumab) Janssen Biotech ulcerative colitis Stelara ® (ustekinumab) Janssen Biotech Crohn's disease tralokinumab AstraZeneca, MedImmune ulcerative colitis Tysabri ® (natalizumab) Biogen Idec Crohn's disease vedolizumab (MLN0002) Takeda Pharmaceuticals Crohn's disease, ulcerative colitis Autoimmune disorders ABT-122 AbbVie rheumatoid arthritis Actemra ® (tocilizumab) Genentech, Roche early rheumatoid arthritis, systemic sclerosis AGS-009 Argos Therapeutics systemic lupus erythematosus (SLE) alemtuzumab Genzyme relapsing-remitting multiple sclerosis AME 527 Applied Molecular rheumatoid arthritis AMG 108 Amgen rheumatoid arthritis AMG 557/MEDI-5872 Amgen, AstraZeneca, MedImmune SLE AMG 714 Amgen rheumatoid arthritis AMG 729 Amgen autoimmune diseases AMG 811 Amgen discoid lupus erythematosus, SLE ART 874 Abbott Laboratories multiple sclerosis anti-CD16 mAb MacroGenics immune thrombocytopenic anti-IL17 mAb (RG7624) Genentech autoimmune disorders anti-LINGO (BIIB033) Biogen Idec Multiple sclerosis Benlysta ® (belimumab) GlaxoSmithKline rheumatoid arthritis, SLE, systemic scleroderma BI-695500 (rituximab biosimilar) Boehringer-Ingelheim rheumatoid arthritis Pharmaceuticals BI-695501 (adalimumab biosimilar) Boehringer-Ingelheim rheumatoid arthritis Pharmaceuticals BT-061 AbbVie, Biotest rheumatoid arthritis Cimzia ® (certolizumab pegol) UCB ankylosing spondylitis, juvenile rheumatoid arthritis clazakizumab (anti-IL6) Bristol-Myers Squibb, Alder rheumatoid arthritis Biopharmaceuticals CNTO-136 (sirukumab) Janssen Biotech rheumatoid arthritis CNTO-1959 Janssen Biotech rheumatoid arthritis daclizumab (anti-CD25 mAb) AbbVie, Biogen Idec multiple sclerosis epratuzumab Immunomedics, UCB SLE ETI-201 Elusys Therapeutics SLE GSK1223249 (NOGO-A mAb) GlaxoSmithKline multiple sclerosis Humira ® (adalimumab) AbbVie rheumatoid arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, psoriasis HuZAF ® (fontolizumab) PDL BioPharma, Biogen Idec rheumatoid arthritis Ilaris ® (canakinumab) Novartis Pharmaceuticals systemic juvenile idiopathic arthritis IMMU-106 (hCD20) Immunomedics autoimmune diseases mavrilimumab AstraZeneca, MedImmune rheumatoid arthritis MEDI-545 (MDX-1103) Medarex, MedImmune lupus MEDI-546 (anti-IFN-alphaR mAb) AstraZeneca, MedImmune SLE MEDI-551 (anti-CD19 mAb) AstraZeneca, MedImmune scleroderma MEDI-570 (anti-ICOS mAb) AstraZeneca, MedImmune SLE MLN 1202 Millennium Pharmaceuticals multiple sclerosis NN8209 (anti-C5aR-151 mAb) Novo Nordisk rheumatoid arthritis NN8210 (anti-C5aR-215 mAb) Novo Nordisk rheumatoid arthritis NN8226 (anti-IL-20 mAb) Novo Nordisk rheumatoid arthritis NN8765 (anti-NKG2 mAb) Novo Nordisk rheumatoid arthritis NN8828 (anti-IL-21 mAb) Novo Nordisk rheumatoid arthritis ocrelizumab (anti-CD20 mAb) Biogen Idec, Genentech, Roche multiple sclerosis, rheumatoid arthritis ofatumumab GlaxoSmithKline multiple sclerosis, rheumatoid arthritis OKT3-gamma-1 Johnson & Johnson psoriatic arthritis olokizumab UCB rheumatoid arthritis otelixizumab (anti-CD3 mAb) GlaxoSmithKline rheumatoid arthritis ozoralizumab (ATN-103) Ablynx rheumatoid arthritis pateclizumab (anti-LT alpha mAb) Genentech rheumatoid arthritis PD-360324 Pfizer cutaneous lupus erythematosus PF-04236921 Pfizer SLE, rheumatoid arthritis PF-05280586 (rituximab biosimilar) Pfizer rheumatoid arthritis Prolia ® (denosumab) Amgen rheumatoid arthritis Remicade ® (infliximab) Janssen Biotech rheumatoid arthritis Rituxan ® (rituximab) Genentech, Biogen Idec rheumatoid arthritis, lupus, primary progressive multiple sclerosis, SLE, relapsing-remitting multiple sclerosis rontalizumab (RG7415) Genentech SLE SAN-300 (anti-VLA-1 antibody) Santams rheumatoid arthritis SAR113244 (anti-CXCR5 mAb) Sanofi US SLE sarilumab (SAR153191) Regeneron Pharmaceuticals, Sanofi rheumatoid arthritis US secukinumab (AIN457) Novartis Pharmaceuticals ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis sifalimumab (anti-IFN-alpha mAb) AstraZeneca, MedImmune SLE Simponi ® (golimumab) Janssen Biotech rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, ankylosing spondylitis, psoriatic arthritis siplizumab (MEDI-507) MedImmune psoriasis Soliris ® (eculizumab) Alexion Pharmaceuticals severe or refractory myasthenia gravis Stelara ® (ustekinumab) Janssen Biotech rheumatoid arthritis, sarcoidosis, plaque psoriasis, multiple sclerosis tabalumab (BAFF inhibitor) Eli Lilly SLE TRX 1 (anti-CD4) TolerRx cutaneous lupus erythematosus TRX 4 TolerRx psoriasis Tysarbi ® (natalizumab) Biogen Idec Multiple sclerosis veltuzumab (IMMU-106) Immunomedics, Takeda immune thrombocytopenic Pharmaceuticals USA purpura, rheumatoid arthritis VX15 Teva Pharmaceuticals, Vaccinex multiple sclerosis XmAb ® 5871 (anti-CD19 mAb) Xencor autoimmune disorders Musculoskeletal Disorders/Arthritis ABT-981 AbbVie osteoarthritis AMG 167 Amgen metabolic bone diseases AMG 745 Amgen muscular atrophy AMG 827 (brodalumab) AstraZeneca, Amgen psoriatic arthritis blosozumab (LY2541546) Eli Lilly osteoporosis BYM338 Novartis Pharmaceuticals sporadic inclusion body myositis, muscular atrophy Cimzia ® (certolizumab pegol) UCB psoriatic arthritis clazakizumab (anti-IL6) Bristol-Myers Squibb, Alder psoriatic arthritis Biopharmaceuticals gevokizumab (IL-1B inhibitor mAb) XOMA osteoarthritis of the hand Humira ® (adalimumab) AbbVie spondylarthritis Ilaris ® (canakinumab) Novartis Pharmaceuticals gouty arthritis ixekizumab (IL-17 antibody) Eli Lilly psoriatic arthritis LY2495655 (anti-myostatin mAb) Eli Lilly disuse muscular atrophy MCS110 Novartis Pharmaceuticals synovitis Prolia ® (denosumab) Amgen male osteoporosis, postmenopausal osteoporosis romosozumab (AMG 785) Amgen postmenopausal osteoporosis SAR391786 (REGN1033) Regeneron Pharmaceuticals, Sanofi treatment of muscle atrophy post- US orthopedic surgery secukinumab (AIN 457) Novartis Pharmaceuticals psoriatic arthritis, polymyalgia rheumatica Stelara ® (ustekinumab) Janssen Biotech psoriatic arthritis tanezumab Pfizer osteoarthritis Cancer and Related Conditions 1311-huA33 Life Science Pharmaceuticals colorectal cancer 1D09C3 GPC Biotech relapsed/refractory B-cell lymphomas 8H9 mAb United Therapeutics metastatic brain cancer 212-Pb-TCMC-trastuzumab AREVA Med HER2-positive cancer metastasized to the abdominal region AbGn-7 AbGenomics International solid tumors ABT-806 AbbVie solid tumors Actimab-A (M195 mAb) Actinium Pharmaceuticals acute myeloid leukemia (AML) Adcetris ® (brentuximab vedotin) Seattle Genetics cutaneous T-cell lymphoma, front- line Hodgkin lymphoma, post- transplant Hodgkin lymphoma relapse prevention, non-Hodgkin lymphoma, non-lymphoma malignancies, CD30-positive hematologic malignancies AGS PSCA mAb Agensys, Merck prostate cancer ALT-836 Altor BioScience, Genentech cancer AME-133v MENTRIK Biotech non-Hodgkin lymphoma AMG 102 Amgen cancer AMG 479 Amgen cancer AMG 623 Amgen B-cell chronic lymphocytic leukemia (CLL) AMG 655 Amgen cancer AMG 706 Amgen imatinib resistant GIST, advanced thyroid cancer AMG 780 Amgen solid tumors AMG 820 Amgen solid tumors AMG 888 (U3-1287) Amgen non-small-cell lung cancer (NSCLC) antibody-drug conjugate (RG7600) Genentech ovarian cancer, pancreatic cancer anti-CD22 ADC (RG7593) Genentech, Seattle Genetics diffuse large B-cell lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL) anti-CD23 MAb Biogen Idec CLL anti-CD45 mAb Actinium Pharmaceuticals AML anti-CD79b ADC (RG7596) Genentech, Seattle Genetics diffuse large B-cell lymphoma, non-Hodgkin lymphoma, CLL anti-CD 80 MAb Biogen Idec non-Hodgkin B-cell lymphoma anti-CXCR4 Bristol-Myers Squibb hematological malignancies anti-EGFL7 mAb (RG7414) Genentech metastatic colorectal cancer, NSCLC, solid tumors anti-FGFR3 mAb (RG7444) Genentech solid tumors anti-HER3/EGFR DAF mAb Genentech colorectal cancer, head and neck (RG7597) cancer anti-idiotype cancer vaccine Viventia Biotech malignant melanoma anti-lymphotoxin beta receptor Biogen Idec solid tumors mAb anti-PD-L1 Bristol-Myers Squibb cancer anti-PD-L1 mAb (RG7446) Genentech melanoma, solid tumors anti-PEM MAb Somanta Pharmaceuticals cancer anti-STEAP1 ADC (RG7450) Genentech, Seattle Genetics prostate cancer anti-Tac(Fv)-E38 immunotoxin National Cancer Institute leukemia, lymphoma APN301 (hu14.18-IL2) Apeiron Biologics malignant melanoma, neuroblastoma in children Archexin ® (RX-0201) Rexahn Pharmaceuticals pancreatic cancer Arzerra ® (ofatumumab) GlaxoSmithKline CLL, diffuse large B-cell lymphoma, follicular lymphoma ASG-5ME Agensys, Seattle Genetics pancreatic cancer, castration- resistant prostate cancer ASG-22ME Agensys, Seattle Genetics solid tumors AV-203 AVEO Oncology solid tumors Avastin ® (bevacizumab) Genentech, Roche ovarian cancer, HER-2 negative- breast cancer, HER-2 positive breast cancer, high-risk carcinoid tumors, glioblastoma multiforme, metastatic ovarian cancer, NSCLC, metastatic colorectal cancer AVE 9633 maytansin-loaded anti- Sanofi Aventis AML CD33 mAb bavituximab Peregrine Pharmaceuticals NSCLC, pancreatic cancer, breast cancer, liver cancer, prostate cancer, rectal adenocarcinoma BAX-69 Baxter International solid tumors BAY 79-4620 Bayer HealthCare Pharmaceuticals solid tumors BAY 94-9343 Bayer HealthCare Pharmaceuticals solid tumors BAY 20-10112 Amgen, Bayer HealthCare solid tumors Pharmaceuticals Bexxar ® (tositumomab) GlaxoSmithKline non-Hodgkin lymphoma BHQ880 Novartis Pharmaceuticals multiple myeloma BI-505 BioInvent International multiple myeloma BI-836845 Boehringer Ingelheim solid tumors Pharmaceuticals bivatuzumab Boehringer Ingelheim cancer Pharmaceuticals blinatumomab Amgen leukemia and lymphoma BrevaRex ™ ViRexx breast cancer, multiple myeloma BT-062 (indatuximab ravtansine) Biotest multiple myeloma BYM338 Novartis Pharmaceuticals cancer-related cachexia Campath ® (alemtuzumab) Berlex Laboratories, Genzyme B-cell chronic lymphocytic leukemia, lymphoma catumaxomab Fresenius Biotech malignant ascites, ovarian cancer CAT 3888 Cambridge Antibody Technology hairy cell leukemia CDX-011 (glembatumumab Celldex Therapeutics breast cancer, malignant melanoma vedotin) CDX-1127 Celldex Therapeutics hematological malignancies, solid tumors CEP-37250/KNK-2804 Teva North America, Kyowa adenocarcinoma Hakko Kirin Pharma ch14.18 mAb United Therapeutics neuroblastoma chimeric mAb National Cancer Institute neuroblastoma Cixutumumab (LY3012217) Eli Lilly, Imclone Systems NSCLC CNTO-328 (siltuximab) Janssen Biotech giant lymph node hyperplasia, multiple myeloma, myelodysplastic syndromes, prostate cancer, renal cancer, Cotara ™ mAb TNT Peregrine Pharmaceuticals recurrent glioblastoma CP-751871 (figitumumab) Pfizer adrenocortical carcinoma, non- small cell lung cancer CS-1008 (tigatuzumab) Daiichi Sankyo pancreatic cancer, colorectal cancer, non-small cell lung cancer, ovarian cancer CSF-1R mAb (IMC-CS4) Eli Lilly solid tumors CT-011 (pidilizumab) CureTech AML, colorectal cancer, diffuse large B-cell lymphoma, follicular lymphoma, malignant melanoma dalotuzumab (MK-0646) Merck breast cancer, neuroendocrine tumors, NSCLC daratumumab Janssen Biotech multiple myeloma DEDN6526A Genentech malignant melanoma demcizumab (OMP-21M18) GlaxoSmithKline, OncoMed solid tumors Pharmaceuticals DFRF4539A Genentech multiple myeloma DI17E6 (anti-integrin mAb) EMD Serono colorectal cancer, prostate cancer DKN-01 Dekkun solid tumors ecromeximab (KW-2871) Life Science Pharmaceuticals metastatic melanoma elotuzumab Bristol-Myers Squibb, AbbVie multiple myeloma EMD 273063 EMD Lexigen solid tumors, malignant melanoma, neuroblastoma, SCLC enavatuzumab AbbVie solid tumors ensituximab (NPC-1C) Neogenix Oncology colorectal cancer, pancreatic cancer epratuzumab Y-90/veltuzumab Immunomedics non-Hodgkin lymphoma combination Erbitux ® (cetuximab) Bristol-Myers Squibb, Eli Lilly, esophageal cancer, colorectal ImClone Systems cancer, squamous cell cancer of the head and neck farletuzumab (MORAb-003) Eisai platinum-sensitive ovarian cancer, NSCLC FG-3019 FibroGen pancreatic cancer ficlatuzumab AVEO Oncology glioblastoma, lymphoma, multiple myeloma, solid tumors flanvotumab (TYRP1 protein) Eli Lilly malignant melanoma Fzd7 (vantictumab) Bayer HealthCare Pharmaceuticals, solid tumors OncoMed Pharmaceuticals ganitumab Amgen pancreatic cancer, breast cancer, colorectal cancer, sarcoma GC-33/RG7686 Chugai Pharma USA, Roche liver cancer GMK Progenies Pharmaceuticals prevention of recurrence following surgery to remove primacy melanoma in high-risk patients GS-6624 (simtuzumab) Gilead Sciences colorectal cancer, pancreatic cancer HCD122 (anti-CD40 mAb) Novartis Pharmaceuticals, XOMA lymphoma Herceptin ® (trastuzumab) Genentech HER2-overexpressing early stage or metastatic breast cancer HGS-ETR1 (mapatumumab) GlaxoSmithKline liver cancer, multiple myeloma, NSCLC, hematologic and solid tumors HGS-TR2J Human Genome Sciences advanced solid tumors HuC242-DM4 ImmunoGen colorectal, gastrointestinal, NSCLC, pancreatic cancers HuL2G7 Galaxy Biotech solid tumors HuMax-CD4 (zanolimumab) Genmab, Serono cutaneous T-cell lymphoma, non- cutaneous T-cell lymphoma HuMax CD20 (ofatumumab) Genmab CLL, non-Hodgkin lymphoma HuMax-EGFr Genmab head and neck cancer HuM195/rGel Targa Therapeutics AML, CML, myelodysplastic syndromes huN901-DM1 (lorvotuzumab ImmunoGen SCLC, multiple myeloma, solid mertansine) tumors icrucumab (LY3012212) Eli Lilly, ImClone Systems bladder cancer, breast cancer, colorectal cancer IMC-TR1 (LY3022859) Eli Lilly, ImClone Systems solid tumors IMGN529 ImmunoGen non-Hodgkin lymphoma IMGN853 ImmunoGen solid tumors IMMU-102 (epratuzumab Y-90) Immunomedics non-Hodgkin lymphoma inotuzumab ozogamicin (CMC-544) Pfizer, UCB aggressive non-Hodgkin lymphoma, ALL Iomab-B (anti-CD45 mAb) Actinium Pharmaceuticals AML J 591 Lu-177 BZL Biologics prostate cancer KB004 KaloBios Pharmaceuticals hematological malignancies LFA102 Novartis Pharmaceuticals, XOMA breast cancer, prostate cancer lirilumab (anti-KIR) Bristol-Myers Squibb cancer LY2495655 (anti-myostatin mAb) Eli Lilly cancer cachexia LY2875358 (c-met mAb) Eli Lilly cancer M195-bismuth 213 conjugate Actinium Pharmaceuticals AML M200 (volociximab) PDL BioPharma, Biogen Idec advanced solid tumors MAb HeFi-1 National Cancer Institute lymphoma, non-Hodgkin lymphoma MABp1 XBiotech cancer-related cachexia, advanced cancer, leukemia MDX-060 (iratumumab) Medarex Hodgkin's disease, anaplastic large- cell-lymphoma MDX-070 Medarex prostate cancer MDX-214 Medarex EGFR-expressing cancers MEDI-522 MedImmune T-cell lymphoma, melanoma, prostate cancer, solid tumors MEDI-551 (anti-CD19 mAb) AstraZeneca, MedImmune hematological malignancies MEDI-573 (anti-IGF mAb) AstraZeneca, MedImmune solid tumors MEDI-575 (anti-PDGFRα mAb) AstraZeneca, MedImmune glioblastoma, NSCLC MEDI-0639 (anti-DLL-4 mAb) AstraZeneca, MedImmune solid tumors MEDI-3617 (anti-ANG-2 mAb) AstraZeneca, MedImmune solid tumors MEDI-4736 (anti-CD274 mAb) AstraZeneca, MedImmune cancer MEDI-6469 (anti-OX40 mAb) AgonOx, AstraZeneca, solid tumors MedImmune MGA271 (anti-B7-H3) MacroGenics solid tumors MGAH22 (anti-HER2) MacroGenics solid tumors milatuzumab Immunomedics CLL milatuzumab-DOX Immunomedics multiple myeloma MINT1526A Genentech solid tumors MK-3475 Merck malignant melanoma, NSCLC MLN0264 (GCC antibody drug Millennium Pharmaceuticals gastrointestinal cancer conjugate) mogamulizimab Kyowa Hakko Kirin Pharma cutaneous T-cell lymphoma, adult T-cell lymphoma, T-cell leukemia MORAb 003 Eisai ovarian cancer MORAb-004 Eisai colorectal cancer, melanoma, sarcoma MORAb-009 (amatuximab) Eisai mesothelioma moxetumomab pasudotox AstraZeneca, MedImmune hematological malignancies Mylotarg ™ (gemtuzumab Wyeth acute myeloid leukemia ozogamicin) necitumumab Bristol-Myers Squibb, Eli Lilly, NSCLC ImClone Systems neuradiab Bradmer Pharmaceuticals glioblastoma nimotuzumab InnoMab PIE glioma, squamous cell carcinomas of the head and neck, recurrent or refractory high grade malignant glioma, anaplastic astrocytomas, glioblastomas and diffuse intrinsic pontine glioma nivolumab (anti-PD1) Bristol-Myers Squibb melanoma, NSCLC, renal cell carcinoma, solid tumors obinutuzumab (GA101) Biogen Idec, Genentech CLL, diffuse large B-cell lymphoma, non-Hodgkin lymphoma olamtumab (LY3012207) Eli Lilly, ImClone Systems glioblastoma OMP-52M51 (anti-Notch 1) GlaxoSmithKline, OncoMed hematological malignancies Pharmaceuticals OMP-59R5 (anti-Notch 2/3) GlaxoSmithKline, OncoMed pancreatic cancer Pharmaceuticals onartuzumab (anti-c-met- mAb) Genentech metastatic NSCLC oregovomab Quest Pharmatech ovarian cancer PAM 4 Merck pancreatic cancer panitumumab (rIIuMAb EGFr) Abgenix colorectal cancer Perjeta ™ (pertuzumab) Genentech early HER2-positive breast cancer, HER2-positive metastatic breast cancer, HER2-positive gastric cancer, ovarian cancer PF-03446962 Pfizer solid tumors PF-04605412 Pfizer solid tumors PF-05082566 Pfizer cancer, lymphoma PF-05280014 (trastuzumab biosimilar) Pfizer metastatic breast cancer PSMA-ADC Progenics Pharmaceuticals prostate cancer R1550 RadioTheraCIM Roche, YM BioSciences metastatic breast cancer, glioma mmucirumab (LY3009806) Eli Lilly, ImClone Systems breast cancer, colorectal cancer, gastric cancer RAV 12 Raven Biotechnologies cancer Redectane ® (girentuximab I-124) Wilex AG diagnosis of kidney cancer REGN1400 Regeneron Pharmaceuticals cancer Rencarex ® G250 Wilex AG renal cancer RG7116 Roche solid tumors RG7155 Roche solid tumors RG7160 (humAb EGFR) Roche colorectal cancer RG7212 Roche solid tumors RG7356 (anti-CD44 mAb) Roche AML, solid tumors RG7458 (antibody-drug conjugate) Genentech, Seattle Genetics ovarian cancer RIGScan ™ Navidea Biopharmaceuticals diagnosis of colorectal cancer rilotumumab Amgen colorectal cancer, gastric cancer, prostate cancer, SCLC Rituxan ® (rituximab) Genentech diffuse large B-cell lymphoma, B- cell non-Hodgkin lymphoma, indolent non-Hodgkin lymphoma induction therapy, relapsed or refractory CLL RON8 mAb Eli Lilly, ImClone Systems cancer SAR3419 (maytansin-loaded anti- Sanofi US ALL, non-Hodgkin lymphoma CD19 mAb) SAR153192 (REGN 421) Regeneron Pharmaceuticals, Sanofi cancer (anti-DLL4 mAb) US SAR256212 (MM-121) Merrimack Pharmaceuticals, Sanofi breast cancer, solid tumors (anti-ErbB3 mAb) US SAR307746 (REGN910) Regeneron Pharmaceuticals, Sanofi solid tumors (anti-angiopoietin-2 mAb) US SAR566658 Sanofi US DS6-positive solid tumors (maytansin-loaded anti-D56) SAR650984 Sanofi US hematological malignancies (anti-CD38 naked mAb) SGN30 Seattle Genetics cutaneous anaplastic large-cell lymphoma, systemic anaplastic large-cell lymphoma, Hodgkin's disease SGN-33 (lintuzumab) Seattle Genetics AML, myelodysplastic syndromes CLL multiple myeloma, non Hodgkin lymphoma SGN-40 Seattle Genetics AML, myelodysplastic syndromes CLL multiple myeloma, non Hodgkin lymphoma SGN-75 (vorsetuzumab mafodotin) Seattle Genetics non-Hodgkin lymphoma, renal cancer sibroturtumab Life Science Pharmaceuticals colorectal, head and neck, lung cancers Sym004 EMD Serono, Symphogen head and neck cancer, solid tumors tabalumab (BAFF inhibitor) Eli Lilly multiple myeloma Tarvacin ™ (bavituximab) Peregrine Pharmaceuticals solid tumors TF2 Immunomedics diagnosis of colorectal cancer TG-1101 (ublituximab) TG Therapeutics CLL, non-Hodgkin lymphoma tigatuzumab Daiichi Sankyo breast cancer, liver cancer, ovarian cancer, pancreatic cancer TNX-650 Tanox refractory Hodgkin lymphoma trastuzumab emtansine (T-DM1) Genentech, Roche HER2-positive metastatic breast cancer, early HER2-positive breast cancer, advanced HER2-positive gastric cancer TRC105 TRACON Pharmaceuticals bladder cancer, liver cancer, ovarian cancer, prostate cancer, solid tumors tremelimumab (anti-CTLA4 mAb) AstraZeneca, MedImmune solid tumors, metastatic melanoma, prostate cancer tumor immunotherapy mAb Genentech solid tumors U3-1565 Daiichi Sankyo solid tumors urelumab (anti-CD137) Bristol-Myers Squibb cancer VAY736 MorphoSys, Novartis CLL Pharmaceuticals VB4-845 Viventia Biotechnologies bladder cancer Vectibix ® (panitumumab) Amgen colorectal cancer VEGFR3 mAb (IMC-3C5) Eli Lilly, ImClone Systems cancer veltuzumab (IMMU-106) Immunomedics CLL, non-Hodgkin lymphoma VGX-100 Circadian Technologies solid tumors volociximab AbbVie NSCLC VX15 Vaccinex solid tumors Xgeva ® (denosumab) Amgen delay or prevention of bone, metastases in prostate cancer or breast cancer, giant cell tumor of the bone XmAb ® (high ADCC mAb) Boehringer Ingelheim cancer Pharmaceuticals, Xencor XmAb ® 2513 (anti-CD30 mAb) Xencor Hodgkin disease, T-cell lymphoma XmAb ® 5574 (anti-CD19 mAb) MorphoSys, Xencor CLL Y-90 hPAM 4 (IMMU-107) Immunomedics pancreatic cancer Yervoy ™ (ipilimumab) Bristol-Myers Squibb adjuvant melanoma, NSCLC, prostate cancer, SCLC, gastric cancer, ovarian cancer, leukemia, lymphoma, renal cell cancer zanolimumab Emergent BioSolutions peripheral T-cell lymphoma Zevalin ® (ibritumomab tiuxetan) Spectrum Pharmaceuticals diffuse large B-cell lymphoma, non-Hodgkin lymphoma Infectious diseases ABthrax ™ (raxibacumab) Human Genome Sciences anthrax Anthim ™ (ETI-204) Elusys Therapeutics anthrax anthrax immune globulin Cangene anthrax anti-HIV-1 mAb Polymun Scientific HIV infection anti-hsp90 mAb NeuTec Pharma candidiasis anti-PD-L1 Bristol-Myers Squibb hepatitis B anti-staph mAb MedImmune prevention of staphylococcal infections Aurexis (tefibazumab) Inhibitex prevention and treatment of S. aureus bacteremia bavituximab Peregrine Pharmaceuticals hepatitis C CCR5 MAb Hunan Genome Sciences HIV infection Cytolin ® (anti-CD8 mAb) CytoDyn HIV infection FGI-101-1A6 Functional Genetics influenza foravirumab Crucell post-exposure prevention of rabies ibalizumab (TMB-355) TaiMed Biologics USA HIV-1 infection KB001-A (antibody-fragment product) KaloBios Pharmaceuticals, Sanofi Pseudomonas infections in cystic Pasteur fibrosis patients, prevention of ventilator-associated pneumonia KD-247 Kaketsuken HIV-1 infection MBL-HCV1 MassBiologics Hepatitis C MDX-066 (CDA-1) Medarex C. difficile disease MDX-1303 Medarex, PharmAthene anthrax MEDI-557 (RSV mAb-extended AstraZeneca, MedImmune prevention of respiratory syncytial half-life) virus (RSV) infections MK-3415A Merck Clostridium difficile infections (actoxumab/bezlotoxumab) NM01 SRD Pharmaceuticals HIV infection Numax ™ (motavizumab) MedImmune RSV PRO 140 CytoDyn HIV-1 infection SAR279356 (anti-PNAG mAB) Sanofi US prevention of bacterial infections Soliris ® (eculizumab) Alexion Pharmaceuticals Shiga toxin E. coli-related hemolytic uremic syndrome (STEC-HUS) streptococcal B vaccine conjugate Novartis Vaccines prevention of streptococcal B infections Synagis ® (palivizumab) MedImmune prevention of RSV infections Tarvacin ™ Peregrine Pharmaceuticals hepatitis C TCN-032 (IgG mAb) Theraclone Sciences influenza A virus infections TCN-202 Theraclone Sciences CMV infections Thravixa ™ (fully human anthrax mAb) Emergent Bio Solutions post-exposure treatment of anthrax TNX-355 Tanox HIV infection UB-421 United Biomedical HIV-1 infection XOMA 3AB XOMA, National Institute of botulism Allergy and Infectious Diseases XTL 6865 XTL Biopharmaceuticals hepatitis C Blood disorders afelimomab Abbot Laboratories sepsis, septic shock Benlysta ® (belimumab) GlaxoSmithKline vasculitis BI-655075 Boehringer Ingelheim blood coagulation disorders Pharmaceuticals eculizumab Alexion Pharmaceuticals paroxysmal nocturnal hemoglobinurea ferroportin mAb Eli Lilly anemia hepcidin mAb Eli Lilly anemia ReoPro ® (abciximab) Eli Lilly adjunct to percutaneous coronary intervention for the prevention of cardiac ischemic complications SelG1 Selexys Pharmaceuticals sickle cell anemia urtoxazumab Teijin Pharma hemolytic uremic Cardiovascular disease AMG 145 Amgen Hypercholesterolemia, hyperlipoproteinemia type IIa anti-fibrin mAb (3B6/22 Tc-99m) Agenix diagnosis of deep vein thrombosis, diagnosis of pulmonary embolism anti-oxLDL (BI-204/RG7418) BioInvent International, Genentech atherosclerosis anti-PCSK9 mAb (RG7652) Genentech cardiovascular disease GSK249320 GlaxoSmithKline stroke IL-1β antibody Eli Lilly cardiovascular disease Ilaris ® (canakinumab) Novartis Pharmaceuticals secondary prevention of cardiovascular events inclacumab (RG1512) Roche peripheral vascular disease MABp1 XBiotech vascular restinosis MLN 1202 Millennium Pharmaceuticals atherosclerosis pexelizumab Alexion Pharmaceuticals, acute myocardial infarction, Procter & Gamble Pharmaceuticals cardiopulmonary bypass PF-04950615 (RN316) Pfizer hypercholesterolemia SAR236553/REGN727 Regeneron Pharmaceuticals, Sanofi hypercholesterolemia (anti-PCSK-9 mAb) US Diabetes and Related Conditions anti-CD3 mAb MacroGenics type 1 diabetes gevokizumab (IL-1B inhibitor mAb) XOMA type 1 diabetes, type 2 diabetes GSK1070806 (IL-18 mAb) GlaxoSmithKline type 2 diabetes Ilaris ® (canakinumab) Novartis Pharmaceuticals type 1 diabetes, type 2 diabetes MABp1 XBiotech type 2 diabetes OKT3-gamma-1 Johnson & Johnson type 1 diabetes teplizumab MacroGenics type 1 diabetes TRX 4 (anti-CD3) TolerRx type 1 diabetes Eye Conditions anti-factor D (RG7417) Genentech geographic atrophy associated with age-related macular degeneration anti-LINGO (BI1B033) Biogen Idec optic neuritis gevokizumab (IL-1B inhibitor mAb) XOMA intermediate or posterior uveitis GSK933776A (anti-B amyloid mAb) GlaxoSmithKline age-related macular degeneration Humira ® (adalimumab) AbbVie uveitis iSONEP ™ (sonepcizumab) Lpath wet age-related macular degeneration Lucentis ® (ranibizumab) Genentech, Roche age-related macular degeneration PF-04382923 (RN6G) Pfizer age-related macular degeneration secukinumab (AIN457) Alcon Labs, Novartis uveitis Pharmaceuticals Soliris ® (eculizumab) Alexion Pharmaceuticals severe or refractory neuromyelitis optica Genetic Disorders KRN-23 Kyowa Hakko Kirin Pharma X-linked dominant hypophosphatemic rickets Soliris ® (eculizumab) Alexion Pharmaceuticals paroxysmal nocturnal hemoglobinuria (PNH) Neurological Disorders AAB-002 Janssen Alzheimer Alzheimer's disease Immunotherapy, Pfizer AAB-003/PF-05236812 Janssen Alzheimer Alzheimer's disease Immunotherapy, Pfizer ABT-110 AbbVie chronic pain ALD403 Alder Biopharmaceuticals prevention of migraine ATI355 (anti-Nogo-A mAb) Novartis Pharmaceuticals spinal cord injury BAN2401 (amyloid beta-protein inhibitor) BioArctic Neuroscience, Eisai Alzheimer's disease bapineuzumab Janssen Alzheimer Alzheimer's disease Immunotherapy, Pfizer crenezumab (anti-Abeta) Genentech Alzheimer's disease fulranumab Janssen Research & Development cancer pain GSK1223249 (NOGO-A mAb) GlaxoSmithKline amyotrophic lateral sclerosis (ALS) GSK933776A (anti-B amyloid mAb) GlaxoSmithKline Alzheimer's disease LY2951742 (CGRP peptide) Arteaus Therapeutics, Eli Lilly migraine prevention MEDI-5117 (anti-IL-6 mAb) AstraZeneca, MedImmune osteoarthritis pain RG1450 (gantenerumab) Roche prodromal Alzheimer's disease RN-307 (anti-CGRP mAb) Labrys Biologics migraine RN624 Rinat Neuroscience osteoarthritis pain RN1219 Rinat Neuroscience Alzheimer's disease SAR228810 (anti-protofibrillar AB mAb) Sanofi US Alzheimer's disease solanezumab (LY2062430) Eli Lilly Alzheimer's disease tanezumab Pfizer chronic pain Respiratory Disorders ABN 912 Novartis Pharmaceuticals asthma, chronic obstructive pulmonary disorders (COPD) ABX-IL8 Amgen COPD ALT-836 Altor BioScience, Genentech acute lung injury, adult respiratory distress syndrome AMG 157/MEDI-9929 Amgen, AstraZeneca asthma AMG 317 Amgen asthma AMG 761 Amgen asthma AMG 827 (brodalumab) Amgen, AstraZeneca asthma benralizumab AstraZeneca, MedImmune asthma, chronic obstructive pulmonary disease (COPD) Bosatria ™ (mepolizumab) GlaxoSmithKline asthma carlumab Janssen Biotech pulmonary fibrosis CNTO-3157 Janssen Biotech asthma CNTO-5825 Janssen Biotech allergic asthma daclizumab (anti-CD25 MAb) Protein Design Labs, Roche asthma FG-3019 FibroGen idiopathic pulmonary fibrosis GS-6624 (simtuzumab) Gilead Sciences idiopathic pulmonary fibrosis KB003 KaloBios Pharmaceuticals severe asthma MEDI-528 (anti-TL-9 mAb) MedImmune asthma MEDI-4212 (anti-IgE mAb) AstraZeneca, MedImmune asthma MEDI-7814 (anti-C5/C5a mAb) AstraZeneca, MedImmune COPD MEDI-8968 (anti-IL-1R mAb) AstraZeneca, MedImmune COPD mepolizumab (anti-TL5 mAb) GlaxoSmithKline asthma and nasal polyposis QAX576 Novartis Pharmaceuticals asthma, idiopathic pulmonary fibrosis QBX258 Novartis Pharmaceuticals asthma QGE031 Novartis Pharmaceuticals allergic asthma quilizumab (anti-M1 prime mAb) Genentech allergic asthma, allergic rhinitis reslizumab Cephalon asthma, eosinophilic esophagitis RG3637 (lebrikizumab) Genentech, Roche severe asthma SAR156597 (bispecific Sanofi US idiopathic pulmonary fibrosis interleukin-4/interleukin-13 mAb) SAR231893 (anti-IL4 mAb) Regeneron Pharmaceuticals, Sanofi asthma US STX-100 Biogen Idec idiopathic pulmonary fibrosis TNX-832 Tanox respiratory diseases tralokinumab AstraZeneca, MedImmune asthma Xolair ® (omalizumab) Genentech, Novartis pediatric asthma Pharmaceuticals Skin Diseases AbGn-168H AbGenomics International plaque psoriasis AMG 827 (brodalumab) Amgen, AstraZeneca psoriasis BT-061 AbbVie, Biotest plaque psoriasis CNTO-1959 Janssen Biotech plaque psoriasis gevokizumab (IL-1B inhibitor mAb) XOMA acne vulgaris Humira ® (adalimumab) AbbVie hidradenitis suppurativa ixekizumab (IL-17 antibody) Eli Lilly psoriasis MABp1 XBiotech acne, psoriasis MK-3222 (tildrakizumab) Merck plaque psoriasis QGE031 Novartis Pharmaceuticals atopic dermatitis REGN846 Regeneron Pharmaceuticals atopic dermatitis SAR231893 (anti-IL4 mAb) Regeneron Pharmaceuticals, Sanofi atopic dermatitis US secukinumab (AIN457) Novartis Pharmaceuticals plaque psoriasis Xolair ® (omalizumab) Genentech, Novartis chronic idiopathic urticaria Pharmaceuticals Transplantation ASKP-1240 Astellas Pharma US, Kyowa Hakko prevention of organ transplant Kirin Pharma rejection Benlysta ® (belimumab) GlaxoSmithKline immunosuppression ORTHOCLONE OKT ® 3 Ortho Biotech acute kidney transplant rejection, (muromomab-CD3) reversal of heart and liver transplant rejection OKT3-gamma-1 Protein Design Labs, Johnson & renal transplant rejection Johnson Simulect ® (basiliximab) Novartis Pharmaceuticals prevention of renal transplant rejection Soliris ® (eculizumab) Alexion Pharmaceuticals presensitized kidney transplant (acute humoral rejection) TOL101 Tolera Therapeutics prevention of transplant rejection Zenapax ® (daclizumab) Roche prophylaxis of acute kidney transplant rejection Other anti-IL31 Bristol-Myers Squibb immunology anti-TWEAK (BIIB 023) Biogen Idec lupus nephritis BAX-69 Baxter International lupus nephritis CNTO-5 Janssen Biotech, MorphoSys inflammation CNTO-136 (sirukumab) Janssen Biotech lupus nephritis CR 0002 CuraGen kidney inflammation FB 301 CytovanceBiologics, Fountain hypersensitivity (IgE-mediated Biopharma allergic diseases) FG-3019 FibroGen liver fibrosis due to chronic hepatitis B infection fresolimumab (TGFβ antagonist) Genzyme fibrosis GS-6624 (simtuzumab) Gilead Sciences liver fibrosis, myelofibrosis GSK1070806 (anti-interleukin 18 mAb) GlaxoSmithKline metabolic disorders Humira ® (adalimumab) AbbVie interstitial cystitis LY2382770 (TGF-β antibody) Eli Lilly diabetic nephropathy mAb Genentech metabolic disorders mepolizumab (anti-IL5 mAb) GlaxoSmithKline hypereosinophilic syndrome, eosinophlic esophagitis Meth-mAb InterveXion Therapeutics methamfetamine abuse Stelara ® (ustekinumab) Janssen Biotech primary biliary cirrhosis VAY736 MorphoSys, Novartis inflammation Pharmaceuticals Xolair ® (omalizumab) Genentech, Tanox peanut allergy

Non-limiting examples of protein-based or polypeptide-based biologics include cytokines (e.g., interleukins), chemokines, growth factors, blood-production stimulating proteins (e.g., erythropoietin), hormones (e.g., Elonva® (follicle stimulating hormone), growth hormone), enzymes (e.g., Pulmozyme® (dornase alfa)), clotting factors, insulin, albumin, fragments thereof, conservatively modified variants thereof, analogs thereof, and combinations thereof.

Examples of cytokines include, but are not limited to, TNFα, TNF-related weak inducer of apoptosis (TWEAK), osteoprotegerin (OPG), IFN-α, IFN-β, IFN-γ, interleukins (e.g., IL-1α, IL-1β, IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-5, IL-6, soluble IL-6 receptor (sIL-6R), IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-23, and IL-27), adipocytokines (e.g., leptin, adiponectin, resistin, active or total plasminogen activator inhibitor-1 (PAI-1), visfatin, and retinol binding protein 4 (RBP4)), and combinations thereof. In particular embodiments, the interleukin comprises IL-2 such as Proleukin® (aldesleukin; recombinant IL-2).

Examples of chemokines include, but are not limited to, CXCL1/GRO1/GROα, CXCL2/GRO2, CXCL3/GRO3, CXCL4/PF-4, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1, CXCL13/BCA-1, CXCL14/BRAK, CXCL15, CXCL16, CXCL17/DMC, CCL1, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL6/C10, CCL7/MCP-3, CCL8/MCP-2, CCL9/CCL10, CCL11/Eotaxin, CCL12/MCP-5, CCL13/MCP-4, CCL14/HCC-1, CCL15/MIP-5, CCL16/LEC, CCL17/TARC, CCL18/MIP-4, CCL19/MIP-3β, CCL20/MIP-3α, CCL21/SLC, CCL22/MDC, CCL23/MPIF1, CCL24/Eotaxin-2, CCL25/TECK, CCL26/Eotaxin-3, CCL27/CTACK, CCL28/MEC, CL1, CL2, CX₃CL1, and combinations thereof.

Non-limiting examples of growth factors include epidermal growth factor (EGF), heparin-binding epidermal growth factor (HB-EGF), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF; also known as SERPINF1), amphiregulin (AREG; also known as schwannoma-derived growth factor (SDGF)), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β1, TGF-β2, TGF-β3, etc.), endothelin-1 (ET-1), keratinocyte growth factor (KGF; also known as FGF7), bone morphogenetic proteins (e.g., BMP1-BMP15), platelet-derived growth factor (PDGF), nerve growth factor (NGF), β-nerve growth factor ((3-NGF), neurotrophic factors (e.g., brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4 (NT4), etc.), growth differentiation factor-9 (GDF-9), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), myostatin (GDF-8), erythropoietin (EPO), thrombopoietin (TPO), and combinations thereof.

Examples of receptor construct-based or fusion protein-based biologics include, but are not limited to, naturally-occurring receptors linked to an immunoglobulin frame (e.g., Orencia® (abatacept; immunoglobin CTLA-4 fusion protein), Amevive® (alefacept; IgG1 fusion protein), ENBREL® (etanercept; recombinant human TNF-receptor fusion protein), engineered proteins combining two different polypeptide species (e.g., Ontak® (denileukin diftitox; engineered protein comprising interleukin-2 and diphtheria toxin), and combinations thereof.

The present invention can therefore be used in methods for detecting and measuring the presence or level of a biologic in a sample from a subject receiving biologic therapy for one or more of the diseases or disorders referred to herein and Table 1, including one or more of the following:

Inflammatory diseases, such as inflammatory bowel disease (IBD) (e.g., Crohn's disease (CD) and ulcerative colitis (UC)), uveitis, sarcoidosis, Wegener's granulomatosis, and other diseases with inflammation as a central feature;

Autoimmune diseases, such as rheumatoid arthritis (RA), multiple scleorisis (MS), systemic lupus erythematosus (SLE), ankylosing spondylitis (Bechterew's disease), lupus, psoriatic arthritis, juvenile idiopathic arthritis, psoriasis, erythematosus, and celiac disease;

Cancer, such as digestive and gastrointestinal cancers (e.g., colorectal cancer, small intestine (small bowel) cancer; gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, gastric (stomach) cancer; esophageal cancer; appendix cancer; and the like); gallbladder cancer; liver cancer; pancreatic cancer; breast cancer; lung cancer (e.g., non-small cell lung cancer); prostate cancer; ovarian cancer; renal cancer (e.g., renal cell carcinoma); cancer of the central nervous system; skin cancer; choriocarcinomas; head and neck cancers; hematological malignancies (e.g., leukemia, lymphoma such as B-cell non-Hodgkin's lymphoma); osteogenic sarcomas (e.g., Ewing sarcoma); soft tissue sarcomas (e.g., Dermatofibrosarcoma Protuberans (DFSP), rhabdomyosarcoma); other soft tissue malignancies, and papillary thyroid carcinomas;

Infectious diseases, such as C. difficile disease, respiratory syncytial virus (RSV), HIV, anthrax, candidiasis, staphylococcal infections, and hepatitis C;

Blood disorders, such as sepsis, septic shock, paroxysmal nocturnal hemoglobinuria, and hemolytic uremic syndrome;

Cardiovascular disease, such as atherosclerosis, acute myocardial infarction, cardiopulmonary bypass, and angina;

Metabolic disorders, such as diabetes, e.g., type 1 diabetes mellitus and type 2 diabetes;

Genetic disorders, such as paroxysmal nocturnal hemoglobinuria (PNH);

Neurological disorders, such as osteoarthritis pain and Alzheimer's disease;

Respiratory disorders, such as asthma, chronic obstructive pulmonary disorders (COPD), nasal polyposis, and pediatric asthma;

Skin diseases, such as psoriasis, including chronic moderate to severe plaque psoriasis;

Transplant rejection, such as acute kidney transplant rejection, reversal of heart and liver transplant rejection, prevention of renal transplant rejection, prophylaxis of acute kidney transplant rejection, and renal transplant rejection; and/or

Other disorders, such as kidney inflammation, postmenopausal osteoporosis (bone disorders), hypereosinophilic syndrome, eosinophilic esophagitis, and peanut allergy.

In particular embodiments, the subject has an inflammatory disease (e.g., inflammatory bowel disease (IBD) such as Crohn's disease (CD) or ulcerative colitis (UC)) or an autoimmune disease (e.g., rheumatoid arthritis).

VIII. Examples

The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results.

Example 1

This example illustrates the validation of a homogeneous mobility shift assay (HMSA) for the measurement of vedolizumab (VLM) and anti-VLM antibodies (ATV) in inflammatory bowel disease (IBD) patient serum.

Vedolizumab (VLM), an α4β7 integrin antagonist, is a therapeutic monoclonal antibody recently approved for use in moderate to severe ulcerative colitis or Crohn's disease patients that have failed to demonstrate adequate response to conventional therapies or TNF alpha antagonists. α4β7 integrin is a gut-specific heterodimeric glycoprotein that is important for leukocyte homing to sites of inflammation within the intestine, via its interaction with mucosal addressin cell adhesion molecule-1 (MadCam-1) expressed on the intestinal vascular endothelium. Availability of diagnostic tests to accurately measure VLM drug levels and anti-VLM antibodies is necessary for the effective use of this novel therapeutic in IBD patients. In succession with our anti-TNF alpha therapeutic drug monitoring assays, we have now developed and validated assays to measure VLM and anti-VLM levels in patient serum.

Methods

Soluble α4β7 protein heterodimer was expressed and purified from mammalian cells (e.g., CHO cells). In particular, the integrin α4 subunit was truncated after the thigh domain (α4Δ620) and integrin β7 after the I-EGF 1 domain ((374527) (see, FIG. 13). Acidic/Basic α-helical coiled coil peptides containing one disulfide-bridge forming Cys residue were attached to the C-terminus of the subunits to stabilize the heterodimer (Takagi et al., Embo J., 18:4607-4615 (2003); O'Shea et al., Curr. Biol., 3:658-667 (1993)). A hexahistidine tag was fused to the C-terminus of the basic peptide to facilitate purification. This recombinant heterodimer was used in a “competition” based homogeneous mobility shift assay (HMSA) format to measure VLM levels in sera of patients on VLM therapy. Patient serum was combined with α4β7 to allow therapeutic VLM to bind with α4β7. Subsequently, fluorescently labeled VLM competed with unlabeled VLM is patient sera for binding to its target, α4β7 followed by separation on HPLC size exclusion chromatography. The amount of “free” VLM-alexa fluor determined VLM levels in patient sera. For the anti-VLM assay, standard curves were created by incubating normal human serum containing known amounts of rabbit anti-VLM antibodies with fluorescently labeled VLM; bound and free VLM were then separated by SEC-HPLC. Method validation was determined according to industry recommendations.

Results

Sensitivity for VLM was 0.35 μg/mL. The lower and upper limits of quantitation (LLOQ and ULOQ) were determined to be 0.625 μg/ml and 14 μg/mL, respectively. For anti-VLM, the LLOQ and ULOQ were 3.13 U/ml and 150 U/ml, respectively. The standard curves generated for each assay showed high reproducibility and sensitivity. Inter- and intra-assay precision showed less than 10% CV and accuracy was within 20%. There was no significant interference from lipemic, hemolyzed, or rheumatoid factor (Rf) serum. See, FIGS. 4-10.

Conclusions

A sensitive and specific assay has been developed and validated to measure VLM and anti-VLM levels in patients undergoing treatment for IBD. The assay format required a unique approach owing to the complexity of a large heterodimeric, heavily glycosylated membrane bound antigen as drug target. Both the drug and anti-drug assays demonstrate high accuracy and precision with high sensitivity with a high tolerance to known interfering agents. The assays are useful for clinical monitoring and drug optimization in individual patients.

Example 2

This example illustrates the validation of a homogeneous mobility shift assay (HMSA) and for the measurement of ustekinumab (UTK) and antibodies-to-ustekinumab (ATU) in inflammatory bowel disease (IBD) patient serum.

Ustekinumab is a therapeutic monoclonal antibody which has potential utility in the treatment of IBD in patients that have failed to respond to conventional therapies or TNF alpha antagonists. Ustekinumab is a monoclonal antibody with specificity for interleukin 12 and interleukin 23 via their common p40 subunit and blocks inflammation through these pathways. Here we describe the analytical validation of a HPLC-based high mobility shift assay to measure UTK as well as antibodies-to-ustekinumab.

Methods

Recombinant, soluble IL12p40 subunit (e.g., SEQ ID NO:7) was expressed and purified from mammalian cells. The assay takes advantage of fluorescently labeled UTK competing with unlabeled UTK in patient sera for binding to IL12p40. After incubation of patient sera with recombinant IL12p40, UTK-alexa fluor 488 was added, before running samples on an HPLC size exclusion column which separates “free” UTK-alexa fluor 488 from UTK-alexa fluor 488 which is bound to IL12p40. The amount of “free” UTK-alexa fluor 488 is a measure of the amount of therapeutic UTK in patient sera. The area under the curve (AUC) of the “free” UTK-alexa fluor 488 is plotted against the log of the UTK concentration in known standard samples and UTK concentration in patient sera are calculated by interpolation. For the ATU assay, standard curves were created by incubating normal human serum containing known amounts of rabbit ATU with fluorescently labeled UTK; bound and free GLM were then separated by SEC-HPLC. Method validation was determined according to industry recommendations.

Results

Sensitivity for UTK was 0.15 μg/ml. The upper limit of quantitation (ULOQ) was determined to be 8 μg/mL and the lower limit of quantitation (LLOQ) was determined to be 0.625 μg/ml. For ATU, the LLOQ and ULOQ were 3.13 U/ml and 150 U/ml respectively. The standard curves generated for each assay showed high reproducibility and sensitivity. Inter- and intra-assay precision showed less than 10% CV and accuracy was within 20%. There was no significant interference from lipemic, hemolyzed, or rheumatoid factor (Rf) serum. See, FIGS. 11-12.

Conclusions

This study describes the validation of a novel “competition” based assay to measure ustekinumab levels in patients undergoing treatment for IBD. The assay format required a unique approach owing to complexity of working with this antigen, due to its tendency to form homodimers and heterodimers in serum making usage of a conventional HMSA assay difficult.

The competition-based HMSA assay showed high accuracy and precision with high sensitivity. In addition, the assay has high tolerance to known interfering agents. These assays are useful for clinical monitoring and drug optimization in individual patients.

Example 3

This example illustrates an exemplary vedolizumab (VLM) competition-based assay methodology of the present invention. One of ordinary skill in the art will appreciate that the assay methodology described in this example is applicable to determining the presence or level of ustekinumab (UTK) as well as other biologics in a sample in situations where the complexity of working with the antigen that binds to the biologic (e.g., the antigen is a membrane-bound protein, a glycosylated protein, a multimeric protein, an insoluble protein, a protein that is difficult to express or purify, and/or a large protein) necessitate the use of a soluble form (e.g., a soluble fragment, variant, or monomer) of the antigen.

Normal human serum (NHS) samples spiked with known amounts of vedolizumab (VLM) are serially diluted. Two-fold serial dilutions starting from 80 μg/ml VLM are diluted in NHS to make 10-point curve (i.e., 80 μg/ml to 0.15625 μg/ml). NHS spiked with 12, 4, and 1 μg/ml VLM are used as positive controls. Standard serum samples, positive controls, and patient samples are added to a 96 well plate. Patient samples are added undiluted or diluted 4× or 8× in NHS to increase the assay dynamic range. Soluble α4β7 antigen and assay diluent are added. The plate is placed on a shaker and allowed to incubate at room temperature for 1 hour. After 1 hour, labeled VLM (e.g., VLM-Alexa Fluor® 488) is added. The plate is again placed on a shaker for a 1 hour incubation. Samples are filtered using a 0.2 μm filter plate. Samples are loaded onto an HPLC autosampler and run sequentially through a size exclusion chromatography column (e.g., a Phenomenex BioSep-SEC-s3000 column) which separates free labeled VLM (e.g., VLM-Alexa Fluor® 488) from labeled VLM bound to the soluble α4β7 antigen.

Software written in R-programming language is used to identify the peak representing free labeled VLM (e.g., VLM-Alexa Fluor® 488) and to determine the area of the peak. The area of this peak gets larger when there is VLM in the patient's sera. By comparing the size of this peak to the standard curve, one can interpolate the patient's VLM levels.

Prism (e.g., GraphPad Prism 6) is used to generate a standard curve by plotting the area of free labeled VLM (e.g., VLM-Alexa Fluor® 488) as a function of serum VLM levels. By comparing the size of this peak to the standard curve, one can interpolate the patient's VLM drug concentration.

The assay described in this example is premised on the competition between the VLM in a sample from a patient receiving VLM therapy and the labeled VLM added to the sample reaction for binding to the soluble α4β7 antigen. The relative ratios of labeled and unlabeled VLM determines how much α4β7 antigen is bound to each and determines the free labeled VLM (e.g., VLM-Alexa Fluor® 488) peak area. The more drug present in the patient sample, the more the labeled VLM remains free as opposed to bound to the α4β7 antigen.

Example 4

This example illustrates the validation of a homogeneous mobility shift assay (HMSA) for the measurement of vedolizumab (VLM) and anti-VLM antibodies in inflammatory bowel disease (IBD) patient serum.

Background and Aims

Vedolizumab, an α4β7 integrin antagonist, is a therapeutic monoclonal antibody recently approved for use in moderate to severe ulcerative colitis and Crohn's disease patients that have failed to demonstrate adequate response to conventional therapies or TNFα antagonists. Availability of diagnostic tests to accurately measure serum VLM and anti-VLM (ATV) levels is necessary for the effective use of this novel therapeutic in IBD patients. Here we describe the analytical validation of the HMSA developed to measure VDM and ATV levels in patient serum as well as its clinical utility.

Methods

Soluble α4β7 heterodimer (e.g., α4Δ620/β7Δ527 heterodimer; see, FIG. 13) was expressed and purified in mammalian cells. This recombinant heterodimer was used in a “competition” based HMSA format to measure serum VLM levels in patients on VLM therapy. Patient serum was combined with α4β7 to allow therapeutic VLM to bind with α4β7. Subsequently, fluorescently labeled VLM competed with unlabeled VLM in patient sera for binding to its target, α4β7, followed by separation on HPLC size exclusion chromatography (see, FIG. 1). For the anti-VLM assay, standard curves were created by incubating normal human serum containing known amounts of rabbit anti-VLM antibodies with fluorescently labeled VLM; bound and free VLM were then separated by SEC-HPLC. Validation was performed according to industry recommendations (Shankar, G., et al. 2008).

Results

VLM and ATV assays show high intra-assay precision and accuracy. Intra-assay precision is less than 10% and intra-assay accuracy is less than 15% error. Run to run, instrument to instrument, and analyst to analyst variability are less than 15% CV and less than 20% error in almost all cases. (Table 2). Sensitivity for VLM was 0.348 μg/mL with a dynamic range of 0.625-14 μg/mL (Table 3). The limit of detection for the ATV assay is <1.56 U/mL. A precise value could not be determined as it is too low to interpolate our curve. The dynamic range for ATV was 3.13-150 U/mL in undiluted serum (Table 3). Normal human serum spiked with VLM or ATV showed good linearity and recovery across serial dilutions (FIGS. 14A and 14B). ATV shows high drug tolerance at levels of 20 μg/mL of VLM (Table 4). Common interfering agents in patient serum were tested and did not interfere at levels seen in IBD patients (FIGS. 15A, 15B and 15C).

TABLE 2 Validation data for accuracy and precision of the assays. Accuracy and Precision of the VLM-HMSA Inter-Assay Precision Intra-Assay Precision Instrument Run to Run Analyst to Analyst to Instrument (n = 10) (n = 10) (n = 5) (n = 10) Hgh Med Low High Med Low High Med Low High Med Low Expected 12 4 1 12 4 1 12 4 1 12 4 1 (μ/mL) Measured 10.34 4.36 0.89 11.22 4.16 1.13 10.51 4.19 1.05 12.30 4.18 1.29 (Mean, U/mL) SD 0.56 0.21 0.10 1.11 0.17 0.18 0.47 0.33 0.16 0.86 0.19 0.08 CV % 5.45 4.82 11.64 9.87 4.07 14.53 4.46 7.97 14.91 6.97 4.44 6.07 Accuracy 13.83 9.07 11.00 6.48 3.91 12.72 12.44 4.76 5.30 2.46 4.44 29.04 (% Error) Accuracy and Precision of the ATV-HMSA Inter-Assay Precision Intra-Assay Precision Instrument Run to Run Analyst to Analyst to Instrument (n = 10) (n = 10) (n = 10) (n = 10) Hgh Med Low High Med Low High Med Low High Med Low Expected 50 25 12.5 50 25 12.5 50 25 12.5 50 25 12.5 (U/mL) Measured 47.31 23.32 11.72 51.53 25.01 13.54 51.52 25.56 13.51 47.31 23.34 11.75 (Mean, U/mL) SD 2.82 1.51 0.79 5.00 3.39 1.76 5.00 2.43 1.86 2.73 1.46 0.78 CV % 5.96 6.46 6.75 9.71 13.56 13.00 9.71 9.52 13.78 5.77 6.25 6.62 Accuracy 5.37 6.71 6.27 7.93 0.04 8.32 3.04 2.25 8.06 5.38 6.65 5.96 (% Error)

TABLE 3 Limits of quantitation for each assay. Lower Upper Limit of Limit of Limit of Limit of Assay Blank Detection Quantitation Quantitation VLM-HMSA n = 30 n = 30 n = 36 n = 36 0.054 μg/mL 0.348 μg/mL 0.625 μg/mL 14 μg/mL ATV-HMSA n = 25 n = 25 n = 36 n = 36 <1.56 U/mL <1.56 U/mL 3.13 U/mL 150 U/mL

TABLE 4 Drug interference in the anti-VLM assay. Vedo [μg/mL] Total ATV [U/mL] 20 51.6 0 49.2 20 26.4 0 24.8 20 13 0 14

Conclusions

A sensitive and specific assay has been developed and validated to measure VLM and anti-VLM levels in patients undergoing treatment for IBD. The assay format required a unique approach owing to the complexity of a large heterodimeric, heavily glycosylated membrane protein as drug target. Both the drug and anti-drug assays demonstrate high accuracy and precision with tolerance to known interfering agents. The development of VLM and anti-VLM assays is useful for clinical monitoring and drug optimization in individual patients.

Example 5

This example illustrates the validation of a homogeneous mobility shift assay (HMSA) for the measurement of ustekinumab (UTK) and antibodies-to-ustekinumab (ATU) in inflammatory bowel disease (IBD) patient serum.

Background and Aims

Ustekinumab is a therapeutic monoclonal antibody which has potential utility in the treatment IBD patients that have failed to respond to conventional therapies or TNFα antagonists. Ustekinumab is specific for IL-12 and IL-23 via their common p40 subunit and blocks inflammation through these pathways. Availability of diagnostic tests to accurately measure serum UST and anti-UST (ATU) levels is necessary for the effective use of this novel therapeutic in IBD patients. Here we describe the analytical validation of a “competition” based HMSA developed to measure UTK levels as well as conventional HMSA to measure ATU levels in patient serum.

Methods

Recombinant, soluble IL12p40 (e.g., SEQ ID NO:7) was expressed and purified from mammalian cells. Recombinant IL12p40 was used in a “competition” based HMSA format to measure serum UTK levels in patients on UTK therapy. Patient serum was combined with rIL12p40 to allow therapeutic UTK to bind with rIL12p40. Subsequently, fluorescently labeled UTK competed with unlabeled UTK in patient sera for binding to its target, rIL12p40, followed by separation on HPLC size exclusion chromatography (see, FIG. 16). For the anti-UTK assay, standard curves were created by incubating normal human serum containing known amounts of rabbit anti-UTK antibodies with fluorescently labeled UTK; bound and free UTK were then separated by SEC-HPLC.

Results

UTK and ATU assays show high intra-assay precision and accuracy. Intra-assay precision is less than 10% and intra-assay accuracy is less than 15% error. Run to run, instrument to instrument, and analyst to analyst variability are less than 15% CV and less than 20% error. (Table 5). Sensitivity for UTK is 0.224 μg/mL with a dynamic range of 0.625-10 μg/mL (Table 6). The limit of detection for the ATU assay is <1.56 U/mL. A precise value could not be determined as it is too low to interpolate from the standard curve. The dynamic range for ATU is 3.13-150 U/mL in undiluted serum (Table 6). Normal human serum spiked with UTK or ATU showed good linearity and recovery across serial dilutions within the assay's dynamic range (FIGS. 17A and 17B). ATU shows high drug tolerance. Levels of 20 μg/mL of UTK do not significantly interfere with ATU detection (Table 7). Common interfering agents in patient serum were tested and did not interfere at levels seen in IBD patients. Only highly hemolyzed serum may potentially interfere but not at levels normally seen in patient sera (FIGS. 18A, 18B and 18C).

TABLE 5 Validation data for precision and accuracy of the assays. Accuracy and Precision of the UTK-HMSA Inter-Assay Precision Intra-Assay Precision Instrument Run to Run Analyst to Analyst to Instrument (n = 5) (n = 5) (n = 5) (n = 5) Hgh Med Low High Med Low High Med Low High Med Low Expected 10 5 2.5 10 5 2.5 10 5 2.5 10 5 2.5 (μg/mL) Measured 8.89 4.52 2.40 10.59 5.03 2.59 10.09 4.93 2.66 10.29 5.26 2.89 (Mean, μg/mL) SD 0.62 0.08 0.05 0.97 0.29 0.13 0.52 0.27 0.18 0.49 0.29 0.09 CV % 6.96 1.76 2.16 9.18 5.67 5.06 5.18 5.39 6.95 4.79 5.44 3.15 Accuracy 11.14 9.55 4.03 5.88 0.69 3.71 0.94 1.42 6.23 2.87 5.28 15.43 (% Error) Accuracy and Precision of the ATU-HMSA Inter-Assay Precision Intra-Assay Precision Instrument Run to Run Analyst to Analyst to Instrument (n = 5) (n5) (n = 5) (n = 5) Hgh Med Low High Med Low High Med Low High Med Low Expected 50 20 10 50 20 10 50 20 10 50 20 10 (U/mL) Measured 49.61 21.08 9.47 50.63 21.51 9.20 51.24 21.48 9.19 49.61 20.39 8.78 (Mean, U/mL) SD 0.33 0.83 0.78 1.16 0.73 0.60 1.80 0.73 0.61 1.85 0.69 0.20 CV % 0.67 3.95 8.27 2.30 3.39 6.52 3.51 3.39 6.60 3.74 3.37 2.30 Accuracy 0.79 5.42 5.30 1.25 7.54 7.96 2.47 7.41 8.11 0.79 1.97 12.23 (% Error)

TABLE 6 Limits of quantitation for each assay. Limits of Quantitation Lower Upper Limit of Limit of Limit of Limit of Assay Blank Detection Quantitation Quantitation UTK-HMSA n = 30 n = 30 n = 36 n = 36 0.077 μg/mL 0.224 μg/mL 0.625 μg/mL 10 μg/mL ATU-HMSA n = 30 n = 30 n = 30 n = 30 Too low to <1.56 U/mL 3.13 U/mL 150 U/mL interpolate

TABLE 7 Drug interference in the anti-UTK assay. UTK [μg/mL] Total ATU [U/mL] 20 58.8 0 51.7 20 16.7 0 22.1 20 5.6 0 9.2

Conclusions

HMSA for UTK and ATU showed high accuracy and precision across a wide dynamic range. The HMSA platform allowed detection of UTK and ATU even in the presence of interfering agents which are known to limit the utility of ELISA/ECLIA methods. The development of a new “competition” based HMSA platform allows for the measurement of therapeutic drug levels when a conventional HMSA is not feasible.

Example 6

This example illustrates experiments performed to improve the dynamic range of the homogeneous mobility shift assay (HMSA) for the measurement of vedolizumab (VDZ).

In an effort to improve the assay's dynamic range, the assay was modified by increasing the amount of VDZ-Alexa488 used in the assay 1.6-fold. Increasing the amount of labeled VDZ and proportionately increasing the amount of α4β7 antigen (e.g., α4Δ620/β7Δ527 heterodimer; see, FIG. 13) increased the dynamic range of the assay. In addition, changing the amount of α4β7 relative to labeled VDZ affected the lower limit of quantification. Excess antigen relative to labeled VDZ made the assay relatively insensitive to drug in patient sera and raised the lower limit of quantification. Conversely, too little antigen relative to labeled VDZ had the effect of lowering the upper limit of quantification. Increasing the amount of labeled VDZ from 75 ng/well to 120 ng/well (1.6-fold) and titrating the antigen such that the presence of antigen binds up 75-80% of the labeled VDZ provided the best compromise between the low-end sensitivity needed as well as an improved dynamic range that would enable the measurement of drug in patient sera without requiring dilutions in most cases.

In addition, the data is plotted such that the area of the VDZ-Alexa488 peak (without dividing by the Blocked-Alexa 488 control peak area) is plotted against the log of the VDZ concentration.

1. Assay Limits Limit of Blank

The Limit of Blank (LOB) was determined from 30 replicates of the standard curve blank. The standard curve blank (negative control) in all assays consisted of 4% normal human serum+40 ng VDZ-Alexa488+165 ng α4β7 per 100 μL injection. The average (mean)+1.645SD of the VDZ-Alexa488 peak area was calculated and then used for calculation of the LOD.

TABLE 8 Assay Limit of Blank (LOB). N 30 Mean (VDZ-Alexa488 peak area) 1.91 SD (VDZ-Alexa488 peak area) 0.09 The average (mean) + 1.645 SD 2.06 Interpolated LOB 0.156/ml* *The average (mean) + 1.645 SD was just below the lowest point of the curve and therefore the lowest point on the curve was taken for the LOB.

Limit of Detection

The Limit of Detection (LOD) was determined by utilizing the measured LOB and replicates of serum containing VDZ at a low concentration which is approaching the LOB. Standard 10 was chosen because it is the lowest point on the curve and nearest the LOB. The LOD was calculated using the equation: LOD=LOB+1.645(SD_(low concentration sample)) (Armbruster et al., 2008). The value was then interpolated from the averaged standard curve of the experiments used in the calculation to yield the concentration in μg/mL.

TABLE 9 Assay Limit of Detection. N 30 Mean (VDZ-Alexa488 peak area) 1.91 SD (VDZ-Alexa488 peak area) 0.061 1.645*SD 0.100 Interpolated (1.645*SD) Too low to interpolate LOD = The average (mean) + 3*SD 0.457 μg/mL

Limit of Quantitation

The Lower Limit of Quantitation (LLOQ) was determined by analyzing interpolated concentrations of 30 replicates of a low concentration VDZ positive sample. In this case, standard 8 (effective serum concentration of 0.625 μg/mL) was chosen. The upper limit of quantitation (ULOQ) was determined by analyzing 30 replicates of a high concentration VDZ positive sample (effective serum concentration equal to 14 μg/mL). LLOQ was defined as the concentration that results in a CV≤20% with Error≤25% and thus measures the assay's precision and accuracy at a low analyte concentration. The ULOQ was also qualified by CV≤20% with Error≤25%.

TABLE 10 LLOQ and ULOQ. LLOQ ULOQ N 30 30 Expected (μg/mL) 1 25 Mean (μg/mL) 1.05 20.95 SD (μg/mL) 0.12 1.44 CV (%) 11.39 6.86 Error (%) 5.13 −16.19 These criteria resulted in the following values for the Assay Limits:

TABLE 11 Assays Limits of Quantitation. LOD 0.457 μg/mL LLOQ    1 μg/mL ULOQ   25 μg/mL

2. Interference Antibody to Vedolizumab (ATV) Interference

In this experiment, 2.5, 5, 10 and 20 μg/mL VDZ was added to either normal human serum or various concentrations of rabbit ATV positive serum. These samples were then analyzed using the assay and the % recovery calculated.

TABLE 12 ATV Interference. Measured VDZ VDZ (μg/mL) spike Control % Recovery (μg/mL) (No ATV) 0 U/mL 3.13 U/mL 6.25 U/mL 12.5 U/mL 25 U/mL 50 U/mL 2.5 2.23 100 90 82 62 0 0 5 4.47 100 93 88 77 60 22 10 9.15 100 91 85 77 71 55 20 17.53 100 87 81 73 69 59

The assay tolerance to ATV is up to 6.25 U/ml in this experiment. The ATV interference is expected as neutralizing ATV will compete with α4β7 for binding to patient VDZ. Only VDZ bound to non-neutralizing ATV is expected to be detected.

Integrin α4β7 Substrate Interference

To test the interference of α4β7, titrations were performed in the range of 1 to 1000 ng/mL. VDZ concentrations were plotted on the intercept of the X-axis correspond to the zero concentration of each interfering agent (FIG. 19).

TABLE 13 α4β7 Interference (100 ng/mL). VDZ Positive High Control Medium Control Low Control Control (12 μg/mL) (4 μg/mL) (1 μg/mL) Mean (μg/mL) 12.57 3.77 1.22 SD (μg/mL) 0.23 0.01 0.04 CV (%) 1.8 0.3 3.5 Recovery (%) 109.6 108.1 106.7

There was no significant interference from levels of α4β7 far exceeding levels one would expect to see in sera from patients being treated with VDZ.

3. Antigen Stability

The stability of α4β7 antigen was assessed through storage at 4 degrees (4° C.) for 1 week. After 1 week, the α4β7 was removed from 4° C. storage and analyzed against a fresh aliquot stored at −70° C. The data from the 4° C. storage samples was then compared to that obtained before storage and the percent error calculated. The aliquots were deemed stable at 4° C. if the error was within 25%.

TABLE 14 Accelerated Stability. Time Point Week 0 Week 1 VDZ Pos. Ctrl High Med Low High Med Low 4° C. Mean (μg/mL) 10.00 3.15 0.88 11.06 4.42 1.12 SD (μg/mL) 0.21 0.45 0.06 0.81 0.21 0.06 CV (%) 2.11 14.24 6.65 7.32 4.81 5.21 Error (%) −16.64 −21.35 −12.34 −7.81 10.50 12.05

Accelerated stability of α4β7. Antigen stored at −70° C. was used in the assay to test VDZ positive controls. Controls came out within specifications (Error≤25% and CV≤25%). Antigen stored at 4° C. was used in the assay to test VDZ positive controls. These controls also came out within specifications. In addition, there was no obvious loss of antigen potency as a result of being stored at 4° C. versus −70° C.

4. Additional Dynamic Range Experiments

Three standard curves were generated using a 1× amount of labeled VDZ (e.g., 75 ng/well of VDZ-Alexa488), as well as 2× and 4× amounts. FIG. 20 shows that proportionately increasing the concentrations of both VDZ-Alexa488 and integrin α4β7 used in the assay increases the assay's dynamic range. It also increases anti-drug antibody tolerance. A maximum LLOQ of 1 μg/mL was the criteria for this assay. In particular, increasing the amount of labeled VDZ and α4β7 by 1.6-fold enabled a maximum LLOQ of 1 μg/mL while providing an improved top-end range of 25 μg/mL (see, Table 11).

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

INFORMAL SEQUENCE LISTING SEQ ID NO: 1 Human alpha 4 integrin fragment MAWEARREPGPRRAAVRETVMLLLCLGVPTGRPYNVDTESALLYQGPHNTLFGYSVVLHSHGANRWLLVG APTANWLANASVINPGAIYRCRIGKNPGQTCEQLQLGSPNGEPCGKTCLEERDNQWLGVTLSRQPGENGS IVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRIAPCYQDYVKKFGENFASCQAGISSFYTKDL IVMGAPGSSYWTGSLFVYNITTNKYKAFLDKQNQVKFGSYLGYSVGAGHFRSQHTTEVVGGAPQHEQIGK AYIFSIDEKELNILHEMKGKKLGSYFGASVCAVDLNADGFSDLLVGAPMQSTIREEGRVFVYINSGSGAV MNAMETNLVGSDKYAARFGESIVNLGDIDNDGFEDVAIGAPQEDDLQGAIYIYNGRADGISSIFSQRIEG LQISKSLSMFGQSISGQIDADNNGYVDVAVGAFRSDSAVLLRIRPVVIVDASLSHPESVNRIKFDCVENG WPSVCIDLTLCFSYKGKEVPGYIVLEYNMSLDVNRKAESPPREYESSNGTSDVITGSIQVSSREANCRTH QAFMRKDVRDILTPIQIEAAYHLGPHVISKRSTEEFPPLQPILQQKKEKDIMKKTINFAR SEQ ID NO: 2 Human beta 7 integrin fragment MVALPMVLVLLLVLSRGESELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPSCAWCKQLNFT ASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGATQLAPQRVRVILRPGEPQQL QVRFLRAEGYPVDLYYLMDLSYSMKDDLERVRQLGHALLVRLQEVIHSVRIGFGSFVDKTVLPFVSTVPS KLRHPCPTRLERCQSPFSFHHVLSLIGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRNV SRLLVFTSDDIFHTAGDGKLGGIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAVIS AALPVYQELSKLIPKSAVGELSEDSSNVVQLIMDAYNSLSSIVTLEHSSLPPGVHISYESQCEGPEKREG KAEDRGQCNHVRINQTVIFWVSLQATHCLPEPHLLRLRALGESEELIVELHTLCDCNCSDIQPQAPHCSD GQGHLQCGVCSCAPGRLGRLCECSVAELSSPDLESGC SEQ ID NO: 3 Human alpha 4 integrin fragment with acidic peptide MAWEARREPGPRRAAVRETVMLLLCLGVPIGRPYNVDTESALLYQGPHNTLFGYSVVLHSHGANRWLLVG APTANWLANASVINPGAIYRCRIGKNPGQICEQLQLGSPNGEPCGKICLEERDNQWLGVILSRQPGENGS IVICGHRWKNIFYIKNENKLPIGGCYGVPPDLRTELSKRIAPCYQDYVKKFGENFASCQAGISSFYIKDL IVMGAPGSSYWIGSLEVYNITINKYKAFLDKQNQVKFGSYLGYSVGAGHERSQHTTEVVGGAPQHEQIGK AYIFSIDEKELNILHEMKGKKLGSYFGASVCAVDLNADGFSDLLVGAPMQSTIREEGRVFVYINSGSGAV MNAMETNLVGSDKYAARFGESIVNLGDIDNDGFEDVAIGAPQEDDLQGAIYIYNGRADGISSIFSQRIEG LQISKSLSMFGQSISGQIDADNNGYVDVAVGAFRSDSAVLLRIRPVVIVDASLSHPESVNRIKFDCVENG WPSVCIDLTLCFSYKGKEVPGYIVLEYNMSLDVNRKAESPPREYESSNGTSDVITGSIQVSSREANCRTH QAFMRKDVRDILTPIQIEAAYHLGPHVISKRSTEEFPPLQPILQQKKEKDIMKKTINFARTGGLAQCEKE LQALEKENAQLEWELQALEKELAQ SEQ ID NO: 4 Human beta 7 integrin fragment with the BASE-p1 sequence of peptide Velcro containing a Cys at the ″d″ positions of the heptad repeat peptide with TEV cleavage site and His6 tag MVALPMVLVLLLVLSRGESELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPSCAWCKQLNFT ASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGATQLAPQRVRVILRPGEPQQL QVRFLRAEGYPVDLYYLMDLSYSMKDDLERVRQLGHALLVRLQEVIHSVRIGFGSFVDKTVLPFVSTVPS KLRHPCPTRLERCQSPFSFHHVLSLIGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRNV SRLLVFTSDDIFHTAGDGKLGGIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAVIS AALPVYQELSKLIPKSAVGELSEDSSNVVQLIMDAYNSLSSIVTLEHSSLPPGVHISYESQCEGPEKREG KAEDRGQCNHVRINQTVIFWVSLQATHCLPEPHLLRLRALGESEELIVELHTLCDCNCSDIQPQAPHCSD GQGHLQCGVCSCAPGRLGRLCECSVAELSSPDLESGCGGLENGYFQGGKNAQCKKKLQALKKKNAQLKWK LQALKKKLAQGGHHHHHH SEQ ID NO: 5 Human IL-12p40 wild-type MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLICDTPEEDGITWILDQSSEVLG SGKILTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRF TCWWLITISIDLIFSVKSSRGSSDPQGVICGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEV MVDAVHKLKYENYISSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLIFCVQVQGK SKREKKDRVFIDKISATVICRKNASISVRAQDRYYSSSWSEWASVPC SEQ ID NO: 6 Human IL-12p40 variant (C199A, C274A) MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLG SGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRF TCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSAAPAAEESLPIEV MVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFAVQVQGK SKREKKDRVFIDKISATVICRKNASISVRAQDRYYSSSWSEWASVPC SEQ ID NO: 7 Human IL-12p40 variant (C199A, C274A) with hexahistidine tag MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLG SGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRF TCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSAAPAAEESLPIEV MVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFAVQVQGK SKREKKDRVFIDKISATVICRKNASISVRAQDRYYSSSWSEWASVPCHHHHHH SEQ ID NO: 8 ACID peptide with cysteine residue (3) AQCEKELQALEKENAQLEWELQALEKELAQ SEQ ID NO: 9 BASE peptide with cysteine residue (16), TEV cleavage site (3-9), and hexahistidine tag (46-51) GGLENGYFQGGKNAQCKKKLQALKKKNAQLKWKLQALKKKLAQGGHHHHHH SEQ ID NO: 10 TEV cleavage site EXXYXQ/S X is any amino acid residue SEQ ID NO: 11 Human IL-12p40 variant (C199S, C274S) MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLG SGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRF TCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEV MVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGK SKREKKDRVFIDKISATVICRKNASISVRAQDRYYSSSWSEWASVPC SEQ ID NO: 12 Human IL-12p40 variant (C199A, C274S) MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLG SGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRF TCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSAAPAAEESLPIEV MVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFSVQVQGK SKREKKDRVFIDKISATVICRKNASISVRAQDRYYSSSWSEWASVPC SEQ ID NO: 13 Human IL-12p40 variant (C199S, C274A) MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLG SGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRF TCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSASPAAEESLPIEV MVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFAVQVQGK SKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSTNSETNASVPC 

1-35. (canceled)
 36. A detection method, comprising: providing a sample from a subject that has been administered a biologic; contacting the sample comprising the biologic with an isolated soluble polypeptide; and indirectly detecting binding of the polypeptide to the biologic in the sample by measuring a form of the biologic used to contact the polypeptide in vitro.
 37. The method of claim 36, wherein the detected binding of the polypeptide to the biologic in the sample determines a presence or amount of the biologic in the sample.
 38. The method of claim 36, further comprising contacting the polypeptide in vitro with the biologic.
 39. The method of claim 38, wherein the biologic used to contact the polypeptide in vitro comprises a label.
 40. The method of claim 39, wherein the label comprises a fluorophore.
 41. The method of claim 36, further comprising comparing the form of the biologic used to contact the polypeptide in vitro to a known amount of the biologic.
 42. The method of claim 36, wherein the sample is serum.
 43. The method of claim 36, wherein the antigen comprises a soluble fragment of a cell surface molecule.
 44. The method of claim 36, wherein the antigen comprises an α4 integrin polypeptide, a β7 integrin polypeptide, or an α4β7 polypeptide comprising the α4 integrin polypeptide and the β7 integrin polypeptide.
 45. The method of claim 44, wherein the an α4 integrin polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1 or SEQ ID NO:3, and the β7 integrin polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2 or SEQ ID NO:4.
 46. The method of claim 44, wherein the α4 integrin polypeptide comprises an ACID peptide.
 47. The method of claim 44, wherein the β7 integrin polypeptide comprises a BASE peptide.
 48. The method of claim 44, wherein the α4 integrin polypeptide or the β7 integrin polypeptide comprises a linker.
 49. The method of claim 44, wherein the α4 integrin polypeptide or the β7 integrin polypeptide comprises an affinity tag.
 50. The method of claim 44, wherein the α4 integrin polypeptide or the β7 integrin polypeptide comprises a protease cleavage site.
 51. The method of claim 44, wherein the biologic is vedolizumab.
 52. The method of claim 36, wherein the antigen comprises a p40 subunit of IL-12 or IL-23.
 53. The method of claim 52, wherein the p40 subunit comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 6, 7, 11, 12, or
 13. 54. The method of claim 52, wherein the antigen further comprises an affinity tag.
 55. The method of claim 52, wherein the biologic is ustekinumab. 